JPWO2004078961A1 - Floating carrier and floating / collecting method - Google Patents

Abstract

A gel-forming floating carrier comprising at least a hydrogel-forming polymer. The floating carrier exhibits a thermoreversible sol-gel transition which is a sol state at a low temperature and a gel state at a high temperature, and is substantially insoluble in water at a high temperature gel state. The sedimentation rate of the iron balls (diameter 4 mm) in the floating carrier is 1 mm / min or less at a temperature 16 ° C. higher than the sol-gel transition temperature and 5 mm / min at a temperature 6 ° C. lower than the sol-gel transition temperature. That's it. Floating capable of floating the suspended matter in a state where application of external force to the suspended matter is limited (for example, without substantially bringing the suspended matter into contact with the container wall that should accommodate the floating carrier) Provided are a carrier and a method for floating and collecting using the carrier.

Description

The present invention relates to a floating carrier capable of floating a floating object in a suitable floating state (that is, a state where application of external force to the floating object is limited).
The use of the suspension carrier of the present invention can be particularly suitably used as a carrier for, for example, animal and plant cell and / or tissue culture (for example, three-dimensional culture or suspension culture of undifferentiated cells).

The usage or application of the floating carrier of the present invention is not particularly limited. For convenience of explanation, the prior art related to the culture of undifferentiated animal and plant cells and / or tissues, which is a recent topic, will be described.
In recent years, liquid culture or suspension culture has been widely used as a method for culturing undifferentiated animals and plants from the viewpoint of easily maintaining an undifferentiated state. In the case of plant cell culture, a technique for isolating protoplasts from plant bodies, culturing the protoplasts, and regenerating and growing callus or hairy roots has been developed. Breeding the body is done. This protoplast culture is mainly carried out in liquid culture. However, liquid culture requires agitation and may cause physical damage such as collision to the protoplast.
In the case of culturing animal cells, embryonic stem cells (ES cells) having the ability to differentiate into all cells of an individual have recently attracted particular attention. For example, mouse ES cells are known to be able to proliferate while maintaining an undifferentiated state in the presence of a leukemia inhibitory factor (LIF) belonging to the interleukin-6 (IL-6) family. Yes. Mouse ES cells can differentiate into all tissues except placenta when injected into blastocysts.
In order to create the same conditions as in vivo in vitro, it is widely performed to form an embryoid body (EB). From EB, differentiation into endoderm, mesoderm, and ectoderm is recognized, and it has been reported that endothelial cells, blood cells, muscle cells, nerve cells, and fat cells can be obtained (Eiji Tsuji et al., Cell Engineering, Vol.20, No.7, 989-993 (2001);
The above-mentioned EB can be obtained by suspending ES cells in a spherical shape without LIF, but it is practically essential that the ES cells are not attached to the vessel wall. This is because when ES cells adhere to the container wall, they differentiate into fibroblast-like adhesive cells.
As described above, the EB can be cultured in a state in which ES cells do not adhere to the vessel wall. A hanging culture (three-dimensional culture is performed by hanging a culture solution containing ES cells on a petri dish ceiling or the like). (hanging culture) method and methylcellulose method in which ES cells are three-dimensionally cultured in a high-viscosity liquid medium containing methylcellulose are known (for details of these hanging culture methods and methylcellulose methods, for example, literature Desbaillets I, etal: Exp Physiol., 85, 645-651 (2000) (non-patent document 2) can be referred to).
In the hanging culture method, suspended cells gather under the drop of the culture solution due to gravity and gradually become spherical, but this method can only make one EB in one drop of culture. It is difficult to create a large amount of EBs. In this hanging culture method, since separate cells usually aggregate to form EB, there is no guarantee that a single ES cell has proliferated to form EB, and contamination of other cells is a problem. Become.
On the other hand, the methyl cellulose method has a problem that ES cells or a cell mass in which ES cells have proliferated gradually settle and adhere to the bottom of the container, and adherent cells such as fibroblasts are generated. Furthermore, since the methylcellulose-containing medium has a high viscosity, there is a problem that it is difficult to seed and mix ES cells and to collect EB.
In addition, in any of the above-described hanging culture method and methylcellulose method, it is difficult to change the medium during the culture by these methods, so it is not possible to supply nutrients or remove waste products, and the growth rate of EB is high. There was a problem of being slow.
Eiji Tsuji et al., Cell Engineering, Vol. 20, no. 7, 989-993 (2001) Desbaillets I, etal: Exp Physiol. 85, 645-651 (2000)

An object of the present invention is to provide a floating carrier that has solved the above-mentioned drawbacks of the prior art, and a floating / collecting method using the carrier.
Another object of the present invention is that the application of external force to the suspended object is limited (for example, without substantially bringing the suspended object into contact with the container wall in which the suspended carrier is to be accommodated). It is an object of the present invention to provide a floating carrier capable of floating the liquid and a floating / recovering method using the carrier.
As a result of diligent research, the present inventor has at least a hydrogel-forming polymer having a sol-gel transition temperature at which a sol is gelled at a lower temperature and gelled at a higher temperature, and the sol-gel transition is thermoreversible. It has been found that the use of a gel-forming composition that is a composition containing a specific iron ball sedimentation rate as a floating carrier is extremely effective for achieving the above object.
The floating carrier of the present invention is based on the above findings, and more specifically, is a gel-forming floating carrier containing at least a hydrogel-forming polymer; the floating carrier is a sol state at a low temperature and a gel at a high temperature. Exhibits a thermoreversible sol-gel transition to a state, and is substantially insoluble in water at a high temperature gel state,
The sedimentation rate of iron balls (diameter 4 mm) in the floating carrier is 1 mm / min or less at a temperature 16 ° C. higher than the sol-gel transition temperature and 5 mm / min at a temperature 6 ° C. lower than the sol-gel transition temperature. It is the above, It is characterized by the above.
According to the present invention, there is further provided a gel-forming floating carrier containing at least a hydrogel-forming polymer; the thermally-reversible sol-gel in which the floating carrier is in a sol state at a low temperature and in a gel state at a high temperature. Exhibit a transition and substantially insoluble in a hot gel state,
There is provided a floating carrier characterized in that the sedimentation rate of iron balls (diameter 4 mm) in the floating carrier is 1 mm / min or less at 37 ° C. and 5 mm / min or more at 10 ° C.
According to the present invention, there is further provided a gel-forming floating carrier comprising at least water and a hydrogel-forming polymer, wherein the floating carrier is in a sol state at a low temperature and in a gel state at a high temperature. The sol-gel transition and substantially insoluble in the high temperature gel state; the sedimentation rate of iron spheres (diameter 4 mm) in the suspended carrier is 16 ° C. higher than the sol-gel transition temperature. Using a floating carrier of 1 mm / min or less and 5 mm / min or more at a temperature 6 ° C. lower than the sol-gel transition temperature;
The suspended carrier is made into a sol state lower than the sol-gel transition temperature, and a suspended matter is added to the suspended carrier,
Hold the suspended object in a gel state higher than the sol-gel transition temperature,
A suspension / recovery method is provided that collects the suspended matter after being held in a sol state lower than the sol-gel transition temperature again.
According to the present invention, a gel-forming floating carrier further comprising at least a hydrogel-forming polymer; a thermoreversible sol-gel transition in which the floating carrier is in a sol state at a low temperature and in a gel state at a high temperature. And exhibit substantially water insolubility in a high temperature gel state, and the sedimentation rate of iron spheres (diameter 4 mm) in the floating carrier is 1 mm / min or less at 37 ° C. and 5 mm / min at 10 ° C. Using a floating carrier that is more than 5 minutes;
The suspended carrier is made into a sol state lower than the sol-gel transition temperature, and a suspended matter is added to the suspended carrier,
Hold the suspended object in a gel state higher than the sol-gel transition temperature,
A floatation / recovery method characterized by recovering the suspended matter after being held again in a sol state lower than the sol-gel transition temperature.
As a result of further research based on the above findings, the present inventors have found that when the above-mentioned floating carrier is used, culture and recovery of biologically derived substances (for example, cells or tissues) may be extremely easy. I found it. For example, it has been found that when the play carrier of the present invention is used, undifferentiated cells such as ES cells can be grown while maintaining the undifferentiated state.
For example, when the floating carrier of the present invention having the above structure is a gel-forming composition containing water, it is undifferentiated because it is in a fluid sol state at a low temperature (a temperature lower than the sol-gel transition temperature). Cells can be seeded and mixed easily. Since this gel-forming composition can be gelated as it is at a high temperature (a temperature higher than the sol-gel transition temperature; for example, a room temperature or a culture temperature of 37 ° C.), stem cells and progenitor cells in the suspension carrier of the present invention. Can be cultured three-dimensionally in the same environment as in vivo.
Furthermore, since the floating carrier of the present invention has an appropriate specific gravity and viscosity in a high-temperature gel state, cells and cell aggregates are not substantially settled in the floating carrier and are prevented from adhering to the bottom surface of the culture vessel. it can. Further surprisingly, in the floating carrier of the present invention, undifferentiated cells such as ES cells can be grown while maintaining an undifferentiated state.
In the floating carrier of the present invention, the hydrogel-forming gel-forming composition is substantially water-insoluble in a gel state at a high temperature (culture temperature). Therefore, a liquid medium is overlaid on the floating carrier of the present invention. (FIG. 1) It is easy to culture cells by suspending the suspension carrier of the present invention in a liquid medium (FIG. 2). When undifferentiated cells proliferate, a large amount of nutrients is required. In the culture method using the floating carrier of the present invention, the necessary nutrients can be supplied from an external liquid medium. In addition, substances that inhibit cell growth, such as waste products generated by cells, can be discharged into an external liquid medium. As a result, in the culture method using the floating carrier of the present invention, cell proliferation can be promoted as compared with the conventional cell culture method.
In order to induce differentiation of undifferentiated cells into target cells, not only cells such as stem cells and progenitor cells but also various chemical mediators such as cell growth factors that promote differentiation and proliferation are usually used. Is necessary. Since the floating carrier of the present invention is gelled at the culture temperature and the hydrogel-forming polymer forms a three-dimensional network structure, these chemical mediators should be retained in the floating carrier for a long period of time. Can do. As a result, good differentiation induction from undifferentiated cells to the target cells is achieved.
In the floating carrier of the present invention, for example, after culturing cells, organs, etc., if cooled again, it returns to a sol state having fluidity at low temperatures, so that the cultured cells, organs, etc. can be easily and without damaging. It can be recovered. In addition, since the floating carrier of the present invention can be easily diluted with water in a sol state lower than its sol-gel transition temperature, it can further increase the fluidity and further facilitate the recovery of cultured cells and organs. be able to.
The above-described floating carrier and floating / collecting method of the present invention include, for example, the following aspects.
[1] A gel-forming floating carrier containing at least a hydrogel-forming polymer, wherein the floating carrier exhibits a thermoreversible sol-gel transition in which it is in a sol state at a low temperature and in a gel state at a high temperature; Show substantially water insolubility in the hot gel state,
The sedimentation rate of iron balls (diameter 4 mm) in the floating carrier is 1 mm / min or less at a temperature 16 ° C. higher than the sol-gel transition temperature and 5 mm / min at a temperature 6 ° C. lower than the sol-gel transition temperature. A floating carrier characterized by the above.
[2] The ratio (b / a) of the sedimentation rate (a) at a temperature 16 ° C. higher than the sol-gel transition temperature to the sedimentation rate (b) at a temperature 6 ° C. lower than the sol-gel transition temperature is 5 or more. The floating carrier according to [1].
[3] A gel-forming floating carrier containing at least a hydrogel-forming polymer, wherein the floating carrier exhibits a thermoreversible sol-gel transition in which it is in a sol state at a low temperature and in a gel state at a high temperature, and Show substantially water insolubility in the hot gel state,
A floating carrier characterized in that the sedimentation rate of iron balls (diameter 4 mm) in the floating carrier is 1 mm / min or less at 37 ° C and 5 mm / min or more at 10 ° C.
[4] The floating carrier according to [3], wherein a ratio (c / d) between a sedimentation rate (c) at 37 ° C. and a sedimentation rate (d) at 10 ° C. is 5 or more.
[5] The floating carrier according to any one of [1] to [4], wherein fibroblasts do not substantially grow in the floating carrier.
[6] The floating carrier according to any one of [1] to [5], wherein the hydrogel-forming polymer is a polymer in which a plurality of blocks having a cloud point and a hydrophilic block are combined.
[7] The floating carrier according to any one of [1] to [6], wherein the sol-gel transition temperature is higher than 0 ° C and lower than 45 ° C.
[8] The floating carrier according to any one of [1] to [7], further comprising water.
[9] The floating carrier according to any one of [1] to [8], further containing a chemical mediator.
[10] A gel-forming floating carrier containing at least water and a hydrogel-forming polymer; a thermoreversible sol-gel transition in which the floating carrier is in a sol state at a low temperature and in a gel state at a high temperature. And substantially insoluble in water in a high temperature gel state; the sedimentation rate of iron spheres (diameter 4 mm) in the suspended carrier is 1 mm / min or less at a temperature 16 ° C. higher than the sol-gel transition temperature. And using a floating carrier that is 5 mm / min or more at a temperature 6 ° C. lower than the sol-gel transition temperature;
The suspended carrier is made into a sol state lower than the sol-gel transition temperature, and a suspended matter is added to the suspended carrier,
Hold the suspended object in a gel state higher than the sol-gel transition temperature,
A floatation / recovery method characterized by recovering the suspended matter after being held again in a sol state lower than the sol-gel transition temperature.
[11] A gel-forming floating carrier comprising at least a hydrogel-forming polymer; the floating carrier exhibits a thermoreversible sol-gel transition in which the floating carrier becomes a sol state at a low temperature and a gel state at a high temperature; The solution is substantially water-insoluble in a high-temperature gel state, and the sedimentation rate of iron balls (diameter 4 mm) in the floating carrier is 1 mm / min or less at 37 ° C. and 5 mm / min or more at 10 ° C. Using a floating carrier;
The suspended carrier is made into a sol state lower than the sol-gel transition temperature, and a suspended matter is added to the suspended carrier,
Hold the suspended object in a gel state higher than the sol-gel transition temperature,
A floatation / recovery method characterized by recovering the suspended matter after being held again in a sol state lower than the sol-gel transition temperature.
[12] The suspension / collection according to [10] or [11], wherein the suspended matter is a cell and / or tissue derived from a living body, and the cell and / or tissue derived from the living body are cultured in a floating carrier Method.
[13] The suspension / collection method according to [12], wherein the suspended matter is an ES cell.
[14] An ES cell cultured and recovered by the method according to [12].
[15] Embryoid body (EB) characterized by being cultured and recovered by the method described in [12] above.

FIG. 1 is a schematic cross-sectional view showing one embodiment (corresponding to Example 2) of the method for culturing cells in the floating carrier of the present invention.
FIG. 2 is a schematic cross-sectional view showing another embodiment of the method for culturing cells in the floating carrier of the present invention.
FIG. 3 is a micrograph (magnification 100 times) after culturing mouse ES cells in the floating carrier of the present invention for 6 days (Example 1).
FIG. 4 is a photomicrograph (magnification 100 times) after culturing mouse ES cells in the suspension carrier of the present invention for 6 days (Example 2), and the layered medium was changed on the third day. .
FIG. 5 is a micrograph (magnification 40 times) after culturing mouse ES cells in a commercially available methylcellulose medium for 6 days (Comparative Example).
FIG. 6 is an alkaline phosphatase-stained image of a mouse ES cell cultured in the suspension carrier of the present invention (with LIF) (100-magnification observation with a phase contrast microscope).
Fig. 7 Alkaline phosphatase-stained image of mouse ES cells cultured in the suspension carrier of the present invention (without LIF) (100-magnification with phase contrast microscope)
Fig. 8 Alkaline phosphatase-stained image of mouse ES cells cultured on feeder cells (with LIF) (100-magnification with phase contrast microscope)
Fig. 9 Alkaline phosphatase-stained image of cynomolgus monkey ES cells cultured in the suspension carrier of the present invention (without LIF) (magnification of 400 times phase contrast microscope)
Fig. 10 Alkaline phosphatase-stained image of cynomolgus monkey ES cells cultured on feeder cells (without LIF) (magnification of phase contrast microscope: 400 times)

Hereinafter, the present invention will be described more specifically with reference to the drawings as necessary. In the following description, “parts” and “%” representing the quantity ratio are based on mass unless otherwise specified.
(Floating carrier)
The floating carrier of the present invention is a gel-forming floating carrier whose aqueous solution contains at least a hydrogel-forming polymer having a sol-gel transition temperature. This gel-forming floating carrier exhibits a thermoreversible sol-gel transition that becomes a sol state at a low temperature and a gel state at a high temperature, and the gel-forming floating carrier is substantially insoluble in water at a high temperature gel state. Show.
Furthermore, the floating carrier of the present invention has an iron ball (diameter 4 mm) sedimentation rate in the carrier of 1 mm / min or less at a temperature 16 ° C. higher than the sol-gel transition temperature, and 6 from the sol-gel transition temperature. It is 5 mm / min or more at a low temperature.
In the present invention, “floating” refers to a solid material in which the suspended substance is to hold or contain the carrier in the carrier (medium) of the invention (for example, the inner wall of a container in which the carrier of the invention is to be contained). It is said to be substantially retained by the carrier of the present invention without substantial contact.
(Sol-gel transition temperature)
In the present invention, the definition and measurement of “sol state”, “gel state” and “sol-gel transition temperature” are described in the literature (H. Yoshioka et al., Journal of Macromolecular Science, A31 (1), 113 (1994)). That is, the dynamic elastic modulus of the sample at an observation frequency of 1 Hz is measured by gradually changing the temperature from the low temperature side to the high temperature side (1 ° C./1 min), and the storage elastic modulus ( The temperature at which G ′, the elastic term) exceeds the loss modulus (G ″, viscosity term) is defined as the sol-gel transition temperature. In general, the state of G ″> G ′ is defined as sol, and the state of G ″ <G ′ is defined as gel. In measuring the sol-gel transition temperature, the following measurement conditions can be preferably used.
<Measuring conditions of dynamic elastic modulus>
Measuring instrument (trade name): Stress-controlled rheometer AR500 (manufactured by TA Instruments)
Concentration of sample solution (or dispersion) (however, as the concentration of “polymer compound having sol-gel transition temperature”): 10 (weight)%
Amount of sample solution: about 0.8 g
Shape and dimensions of measuring cell: acrylic parallel disk (diameter 4.0 cm),
Gap 600μm
Measurement frequency: 1Hz
Applied stress: in the linear region.
A hydrogel having a suitable sol-gel transition temperature can be easily selected according to the screening method (sol-gel transition temperature measurement method) described above from specific compounds as described below. In a series of operations for processing and collecting a suspended matter using the floating carrier of the present invention, the above-described sol-gel transition temperature (e ° C.), the treatment temperature of the suspended matter (f ° C.), and the suspended matter Is preferably set to a temperature (g ° C.) during cooling for dispersing, mixing or recovering. That is, it is preferable that there is a relationship of f>e> g among the above three temperatures e ° C., f ° C., and g ° C. More specifically, (fe) is preferably 1 to 90 ° C., more preferably 2 to 50 ° C., and (eg) is 1 to 50 ° C., further 2 to 40 ° C. Is preferred.
(Aspect used for culturing cells and biological tissues)
In an embodiment in which the floating carrier of the present invention is used for culturing cells and biological tissues, the sol-gel transition temperature is higher than 0 ° C and lower than 45 ° C from the viewpoint of preventing thermal damage of the cells and biological tissues. Further, it is preferably higher than 0 ° C. and 42 ° C. or lower (particularly 4 ° C. or higher and 40 ° C. or lower).
A hydrogel having such a suitable sol-gel transition temperature can be easily selected from the specific compounds described below according to the screening method (sol-gel transition temperature measurement method) described above. In a series of operations for culturing cells using the floating carrier of the present invention, the sol-gel transition temperature (e ° C.) described above is the cell culture temperature (f ° C.) and the cells are seeded, mixed or collected. It is preferable to set between the temperature at the time of cooling (g ° C.). That is, it is preferable that there is a relationship of f>e> g among the above three temperatures e ° C., f ° C., and g ° C. More specifically, (fe) is preferably 1 to 40 ° C., more preferably 2 to 30 ° C., and (eg) is 1 to 40 ° C., more preferably 2 to 30 ° C. Is preferred.
(Settling speed of iron ball)
The floating carrier of the present invention has an appropriate specific gravity and viscosity in order to avoid sedimentation of suspended matter (cells and the like). The fact that the floating carrier of the present invention has such a suitable specific gravity and viscosity can be determined, for example, by measuring the sedimentation rate of iron balls in the floating carrier of the present invention by the following method.
<Measurement of sedimentation velocity of iron ball>
Iron ball (stainless steel ball for ball bearing): Diameter 4mm, Mass: 0.26g
Sample volume: 5 mL
Measuring instrument: Clear glass 5 mL graduated cylinder (1 mL scale interval is approximately 14 mm)
About 5 mL of a sample to be measured (floating carrier) is placed in a 5 mL graduated cylinder made of transparent glass, and left to stand (about 2 hours) until reaching a predetermined temperature in a thermostat. An iron ball (diameter 4 mm, mass: 0.26 g) is gently placed on the sample in the graduated cylinder at a predetermined temperature. While maintaining a predetermined temperature in a thermostat, the sedimentation rate of the iron ball is measured.
More specifically, for example, the time from when the upper end of the iron ball passes the 4 mL scale of the graduated cylinder until the upper end of the iron ball passes the 3 mL scale is measured (T minutes), and the 4 mL scale and 3 mL are measured. The settling velocity (Vmm / min) of the iron ball is obtained by dividing the interval (Dmm).
V = D / T
The floating carrier of the present invention has the above iron ball sedimentation velocity V at a temperature 16 ° C. higher than the sol-gel transition temperature. ₊₁₆ Is 1 mm / min or less. This settling velocity V ₊₁₆ Is preferably 0.1 mm / min or less, more preferably 0.01 mm / min or less, and still more preferably 0.001 mm / min or less. Sedimentation velocity V of the iron ball in the suspended carrier at a temperature 16 ° C. higher than the sol-gel transition temperature V ₊₁₆ If this range is exceeded, the suspended matter tends to settle in the suspended carrier during cell culture in a high-temperature gel state, and the suspended matter tends to adhere to the bottom surface of the container.
In addition, the floating carrier of the present invention has a sedimentation velocity V of the iron ball at a temperature 6 ° C. lower than the sol-gel transition temperature. _-6 Is 5 mm / min or more. The sedimentation rate is preferably 10 mm / min or more, more preferably 50 mm / min or more, and further preferably 500 mm / min or more. Sedimentation velocity V of the iron ball in the suspended carrier at a temperature 6 ° C. lower than the sol-gel transition temperature _-6 When the value is below this range, there is a tendency that dispersion, mixing or recovery of suspended matter in a low-temperature sol state becomes difficult.
In the floating carrier of the present invention, V _-6 / V ₊₁₆ Is preferably 5 or more, more preferably 50 or more, and particularly preferably 500 or more (more preferably 5000 or more).
(Aspect used for biological material)
In an embodiment used for a biological material, the floating carrier of the present invention has a sedimentation velocity V of the iron ball at 37 ° C. ₃₇ Is preferably 1 mm / min or less. This settling velocity V ₃₇ Is more preferably 0.1 mm / min or less, still more preferably 0.01 mm / min or less, and particularly preferably 0.001 mm / min or less. Sedimentation velocity V of the above iron ball in the floating carrier at 37 ° C ₃₇ If this range is exceeded, cells are likely to settle in the floating carrier during cell culture in a high-temperature gel state, and are liable to adhere to the bottom surface of the culture vessel.
In an embodiment used for a biological material, the floating carrier of the present invention has a sedimentation velocity V of the iron ball at 10 ° C. ₁₀ Is preferably 5 mm / min or more, more preferably 10 mm / min or more, still more preferably 50 mm / min or more, and particularly preferably 500 mm / min or more. Sedimentation velocity V of the iron ball in the floating carrier at 10 ° C ₁₀ If it is below this range, cell seeding, mixing or recovery in a low-temperature sol state becomes difficult, which is not preferable.
In the floating carrier of the present invention, V ₁₀ / V ₃₇ Is preferably 5 or more, more preferably 50 or more, particularly preferably 500 or more, and further preferably 5000 or more.
(Adjustment of sedimentation speed)
In order to decrease the sedimentation rate of suspended matter (in the case of solid or liquid; including iron balls) in the suspended carrier of the present invention, 1) increase the specific gravity of the suspended carrier, and 2) increase the concentration of the hydrogel-forming polymer. Means to increase, 3) increase the molecular weight of the hydrogel-forming polymer, 4) lower the sol-gel transition temperature, and 5) increase the crosslinking point of the hydrogel can be used.
On the other hand, in order to increase the sedimentation rate of suspended matter (in the case of solid or liquid; including iron balls) in the suspended carrier of the present invention, 1) lower the specific gravity of the suspended carrier, 2) the hydrogel-forming polymer Means such as decreasing the concentration, 3) decreasing the molecular weight of the hydrogel-forming polymer, 4) increasing the sol-gel transition temperature, and 5) decreasing the crosslinking point of the hydrogel can be used.
(Followability for movement of floating carrier)
The hydrogel based on the floating carrier of the present invention shows a solid behavior at a higher frequency and lower on the other hand in terms of the balance of conformity to the morphological change of the cell clump accompanying the cell proliferation. It is preferable to exhibit a liquid behavior with respect to the frequency. More specifically, the followability to the operation of the hydrogel can be suitably measured by the following method.
(Measuring method for tracking performance)
The floating carrier of the present invention containing a hydrogel-forming polymer (1 mL as a hydrogel) is put in a test tube having an inner diameter of 1 cm in a sol state (temperature lower than the sol-gel transition temperature), and the sol-gel transition of the floating carrier is performed. The test tube is held for 12 hours in a water bath at a temperature sufficiently higher than the temperature (for example, about 10 ° C. higher than the sol-gel transition temperature) to gel the hydrogel.
Next, the time (Tm) until the solution / air interface (meniscus) is deformed by its own weight when the test tube is turned upside down is measured. Here, the hydrogel behaves as a liquid for operation at a frequency lower than 1 / Tm (sec-1), and the hydrogel as a solid for operation at a frequency higher than 1 / Tm (sec-1). Will behave. In the case of the hydrogel of the present invention, Tm is 1 minute to 24 hours, preferably 5 minutes to 10 hours.
(Steady flow viscosity)
The gel-like properties of the hydrogel based on the floating carrier of the present invention can also be suitably measured by measuring the steady flow viscosity. The steady flow viscosity η (eta) can be measured, for example, by a creep experiment.
In the creep experiment, a constant shear stress is applied to the sample and the temporal change of shear strain is observed. Generally, in the creep behavior of a viscoelastic body, the shear rate initially changes with time, but thereafter the shear rate becomes constant. The ratio between the shear stress and the shear rate at this time is defined as the steady flow viscosity η. This steady flow viscosity is sometimes referred to as Newtonian viscosity. Here, however, the steady flow viscosity must be determined within a linear region that is largely independent of shear stress.
A specific measuring method is as follows: a stress-controlled viscoelasticity measuring device CSL type rheometer (CSL500, manufactured by Carrie Med, USA) is used as a measuring device, an acrylic disk (diameter 4 cm) is used as a measuring device, and a sample thickness is 600 μm. Observe the measurement time creep behavior (delay curve) of at least 5 minutes. Sampling time is once per second for the first 100 seconds and then once every 10 seconds.
In determining the applied shear stress (stress), the displacement angle is 2 × 10 by applying the shear stress for 10 seconds. ^-3 Set to the lowest value detected above rad. The analysis employs at least 20 measured values after 5 minutes. The hydrogel based on the floating carrier of the present invention has a η of 5 × 10 at a temperature about 10 ° C. higher than its sol-gel transition temperature. ³ ~ 5x10 ⁶ It is preferably Pa · sec, and more preferably 8 × 10. ³ ~ 2x10 ⁶ Pa · sec, especially 1 × 10 ⁴ Pa · sec or more, 1 × 10 ⁶ It is preferably Pa · sec or less.
The above η is 5 × 10 ³ If it is less than Pa · sec, the fluidity is relatively high even in short-time observation, and the three-dimensional retention of cells and tissues by the gel becomes insufficient, making it difficult to function as a floating carrier. On the other hand, η is 5 × 10 ⁶ When Pa · sec is exceeded, the gel tends to hardly exhibit fluidity even when observed for a long time, and the difficulty of following the movement accompanying the regeneration of the biological tissue increases. Η is 5 × 10 ⁶ If it exceeds 1, the possibility that the gel exhibits brittleness increases, and after a slight pure elastic deformation, the tendency to easily cause brittle fracture that breaks at once increases.
(Dynamic elastic modulus)
The gel property of the hydrogel based on the floating carrier of the present invention can be suitably measured also by the dynamic elastic modulus. The gel has an amplitude γ ₀ , Strain γ (t) = γ with frequency ω / 2π ₀ When cos ωt (t is time) is given, the constant stress is σ ₀ , Σ (t) = σ, where δ is the phase difference ₀ Assume that cos (ωt + δ) is obtained. | G | = σ ₀ / Γ ₀ Then, the ratio (G ″ / G ′) between the dynamic elastic modulus G ′ (ω) = | G | cos δ and the loss elastic modulus G ″ (ω) = | G | sin δ is an index representing gel-like properties. It becomes.
The hydrogel based on the floating carrier of the present invention behaves as a solid with respect to strain of ω / 2π = 1 Hz (corresponding to fast operation) and ω / 2π = 10. ^-4 It behaves as a solid against Hz distortions (corresponding to slow operation). More specifically, the hydrogel based on the floating carrier of the present invention preferably exhibits the following properties (for details of such elastic modulus measurement, see, for example, Document: Ryohei Oda, Modern Industrial Chemistry 19 359, Asakura Shoten, 1985).
When ω / 2π = 1 Hz (frequency at which the gel behaves as a solid), (G ″ / G ′) _s = (Tan δ) _s Is preferably less than 1 (more preferably 0.8 or less, particularly preferably 0.5 or less).
ω / 2π = 10 ^-4 In the case of Hz (frequency at which the gel behaves as a liquid), (G ″ / G ′) _L = (Tan δ) _L Is preferably 1 or more (more preferably 1.5 or more, particularly preferably 2 or more).
Above (tan δ) _s And (tan δ) _L And ratio {(tan δ) _s / (Tan δ) _L } Is preferably less than 1 (more preferably 0.8 or less, particularly preferably 0.5 or less).
<Measurement conditions>
Concentration of hydrogel-forming polymer: about 8% by mass Temperature: Temperature about 10 ° C. higher than the sol-gel transition temperature of the floating carrier Measuring instrument: Stress-controlled rheometer (model name: CSL 500, manufactured by Carrie Med, USA)
(Hydrogel-forming polymer)
As long as the thermoreversible sol-gel transition as described above is exhibited (that is, having a sol-gel transition temperature), the hydrogel-forming polymer that can be used in the floating carrier of the present invention is not particularly limited. For example, the hydrogel-forming polymer can be used from the viewpoint that the carrier used in the biological material, that is, the carrier of the present invention easily exhibits a suitable sol-gel change at a physiological temperature (about 0 to 42 ° C.). It is preferably achieved by adjusting the cloud point of a plurality of blocks having a cloud point in the middle and the hydrophilic block, the composition of both blocks and the hydrophobicity, hydrophilicity, and / or molecular weight of both blocks, respectively. .
Specific examples of the polymer in which the aqueous solution has a sol-gel transition temperature and reversibly shows a sol state at a temperature lower than the transition temperature include, for example, a block copolymer of polypropylene oxide and polyethylene oxide. Known polyalkylene oxide block copolymers; etherified celluloses such as methyl cellulose and hydroxypropyl cellulose; chitosan derivatives (KR Holme. Et al. Macromolecules, 24, 3828 (1991)) and the like are known.
As a polyalkylene oxide block copolymer, a pluronic F-127 (trade name, manufactured by BASF Wyandotte Chemicals Co.) gel in which polyethylene oxide is bonded to both ends of polypropylene oxide has been developed. It is known that this high-concentration aqueous solution of Pluronic F-127 becomes a hydrogel at about 20 ° C. or higher and becomes an aqueous solution at a lower temperature. However, in the case of this material, it becomes a gel state only at a high concentration of about 20% by mass or more, and even if it is kept at a temperature higher than the gelation temperature at a high concentration of about 20% by mass or more, further water is added. And the gel will dissolve. Pluronic F-127 has a relatively small molecular weight and not only exhibits a very high osmotic pressure in a gel state of high concentration of about 20% by mass or more, but also easily permeates the cell membrane, thus adversely affecting cells and tissues. there is a possibility.
On the other hand, in the case of etherified cellulose represented by methyl cellulose, hydroxypropyl cellulose and the like, usually, the sol-gel transition temperature is high and is about 45 ° C. or higher (N. Sarkar, J. Appl. Polym. Science, 24 , 1073, 1979). On the other hand, since the body temperature of the living body is usually around 37 ° C., the etherified cellulose is in a sol state, and it is practically difficult to use the etherified cellulose as the floating carrier of the present invention.
As described above, the problems of the conventional polymer in which the aqueous solution has a sol-gel transition point and reversibly shows a sol state at a temperature lower than the transition temperature are 1) higher than the sol-gel transition temperature. Once gelled at temperature, the gel dissolves when water is further added. 2) The sol-gel transition temperature is higher than the body temperature of the living body (around 37 ° C.), and the body temperature is in the sol state. ) In order to cause gelation, the polymer concentration of the aqueous solution needs to be very high.
On the other hand, according to the study by the present inventors, a hydrogel-forming polymer having a sol-gel transition temperature that is preferably higher than 0 ° C. and not higher than 42 ° C. (for example, a plurality of blocks having cloud points) And a hydrophilic block, and the aqueous solution has a sol-gel transition temperature and a polymer that reversibly shows a sol state at a temperature lower than the sol-gel transition temperature). It has been found that the above problem is solved when a floating carrier is constructed.
(Suitable hydrogel-forming polymer)
The hydrogel-forming polymer utilizing a hydrophobic bond that can be suitably used as the floating carrier of the present invention is preferably formed by bonding a plurality of blocks having a cloud point and a hydrophilic block. The hydrophilic block is preferably present because the hydrogel becomes water-soluble at a temperature lower than the sol-gel transition temperature, and the plurality of blocks having a cloud point have a hydrogel having a sol-gel transition temperature. It is preferably present to change to a gel state at higher temperatures. In other words, a block having a cloud point dissolves in water at a temperature lower than the cloud point and changes to insoluble in water at a temperature higher than the cloud point, so that at a temperature higher than the cloud point, the block has a gel. It serves as a cross-linking point consisting of a hydrophobic bond to form. That is, the cloud point derived from the hydrophobic bond corresponds to the sol-gel transition temperature of the hydrogel.
However, the cloud point and the sol-gel transition temperature do not necessarily match. This is because the cloud point of the “block having a cloud point” described above is generally affected by the bond between the block and the hydrophilic block.
The hydrogel used in the present invention utilizes the property that not only the hydrophobic bond becomes stronger as the temperature increases, but also the change is reversible with respect to temperature. From the viewpoint that a plurality of crosslinking points are formed in one molecule and a gel having excellent stability is formed, the hydrogel-forming polymer preferably has a plurality of “blocks having cloud points”.
On the other hand, the hydrophilic block in the hydrogel-forming polymer has the function of changing the hydrogel-forming polymer to water-soluble at a temperature lower than the sol-gel transition temperature, as described above. The hydrogel has a function of forming a hydrogel state while preventing the hydrogel from aggregating and precipitating at a temperature higher than the transition temperature.
(Multiple blocks with cloud points)
The block having a cloud point is preferably a polymer block having a negative solubility-temperature coefficient in water, and more specifically, polypropylene oxide, a copolymer of propylene oxide and another alkylene oxide, A polymer selected from the group consisting of poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, copolymers of N-substituted acrylamide derivatives and N-substituted methacrylamide derivatives, polyvinyl methyl ether, and polyvinyl alcohol partially acetylated products. Can be preferably used. The above polymer (block having a cloud point) has a cloud point higher than 4 ° C. and not higher than 40 ° C., and the polymer used in the present invention (a compound in which a plurality of blocks having a cloud point and a hydrophilic block are combined) ) To a sol-gel transition temperature of 4 ° C. or higher and 40 ° C. or lower.
Here, the cloud point is measured by, for example, cooling an aqueous solution of about 1% by mass of the above polymer (block having a cloud point) to form a transparent uniform solution, and then gradually increasing the temperature (temperature increase rate of about 1). C./min), and the point at which the solution becomes cloudy for the first time is taken as the cloud point.
Specific examples of poly N-substituted acrylamide derivatives and poly N-substituted methacrylamide derivatives that can be used in the present invention are listed below.
Poly-N-acryloylpiperidine; poly-Nn-propylmethacrylamide; poly-N-isopropylacrylamide; poly-N, N-diethylacrylamide; poly-N-isopropylmethacrylamide; poly-N-cyclopropylacrylamide; -N-acryloylpyrrolidine; poly-N, N-ethylmethylacrylamide; poly-N-cyclopropylmethacrylamide; poly-N-ethylacrylamide.
The polymer may be a homopolymer or a copolymer of a monomer constituting the polymer and another monomer. As another monomer constituting such a copolymer, either a hydrophilic monomer or a hydrophobic monomer can be used. In general, copolymerization with a hydrophilic monomer raises the cloud point of the product and copolymerization with a hydrophobic monomer lowers the cloud point of the product. Therefore, a polymer having a desired cloud point (for example, a cloud point higher than 4 ° C. and lower than 40 ° C.) can be obtained also by selecting the monomer to be copolymerized.
(Hydrophilic monomer)
Examples of the hydrophilic monomer include N-vinyl pyrrolidone, vinyl pyridine, acrylamide, methacrylamide, N-methyl acrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, and acrylic having an acid group. Acid, methacrylic acid and salts thereof, vinyl sulfonic acid, styrene sulfonic acid and the like, and N, N-dimethylaminoethyl methacrylate, N, N-diethylaminoethyl methacrylate, N, N-dimethylaminopropyl having a basic group Examples include, but are not limited to, acrylamide and salts thereof.
(Hydrophobic monomer)
On the other hand, examples of the hydrophobic monomer include acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methyl methacrylate and glycidyl methacrylate, N-substituted alkylmethacrylamide derivatives such as Nn-butylmethacrylamide, vinyl chloride, acrylonitrile and styrene. And vinyl acetate and the like, but are not limited thereto.
(Hydrophilic block)
On the other hand, as the hydrophilic block to be combined with the above-described block having a cloud point, specifically, methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, poly N-vinyl pyrrolidone, polyvinyl pyridine, polyacrylamide, polymethacrylamide , Poly N-methylacrylamide, polyhydroxymethyl acrylate, polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid and their salts; poly N, N-dimethylaminoethyl methacrylate, poly N, N-diethylaminoethyl methacrylate , Poly N, N-dimethylaminopropylacrylamide and salts thereof.
The method for bonding the block having a cloud point and the hydrophilic block is not particularly limited. For example, a polymerizable functional group (for example, acryloyl group) is introduced into one of the blocks to give the other block. This can be done by copolymerizing monomers. Further, the combined product of a block having a cloud point and the hydrophilic block may be obtained by block copolymerization of a monomer that gives a block having a cloud point and a monomer that gives a hydrophilic block. Is possible. In addition, the block having a cloud point and the hydrophilic block are bonded to each other by introducing a reactive functional group (for example, a hydroxyl group, an amino group, a carboxyl group, an isocyanate group, etc.) in advance and bonding them together by a chemical reaction. Can also be done. At this time, usually a plurality of reactive functional groups are introduced into the hydrophilic block. In addition, the bond between the polypropylene oxide having a cloud point and the hydrophilic block is repeated, for example, by anionic polymerization or cationic polymerization by repeatedly repeating propylene oxide and a monomer (for example, ethylene oxide) constituting “another hydrophilic block”. By polymerizing, a block copolymer in which polypropylene oxide and a “hydrophilic block” (for example, polyethylene oxide) are bonded can be obtained. Such a block copolymer can also be obtained by copolymerizing monomers constituting a hydrophilic block after introducing a polymerizable group (for example, acryloyl group) to the terminal of polypropylene oxide. Furthermore, the polymer used in the present invention can also be obtained by introducing a functional group capable of binding reaction with a functional group (for example, hydroxyl group) at the end of polypropylene oxide into the hydrophilic block and reacting both. . Moreover, the hydrogel-forming polymer used in the present invention can also be obtained by linking materials such as Pluronic F-127 (trade name, manufactured by Asahi Denka Kogyo Co., Ltd.) in which polyethylene glycol is bonded to both ends of polypropylene glycol. Can be obtained.
Since the polymer of the present invention in a mode including a block having a cloud point is water-soluble at a temperature lower than the cloud point, the above “block having a cloud point” present in the molecule is water-soluble. It is completely dissolved in water and shows a sol state. However, when the temperature of the polymer aqueous solution is raised to a temperature higher than the above cloud point, the “block having a cloud point” present in the molecule becomes hydrophobic, and is associated between separate molecules by hydrophobic interaction. To do.
On the other hand, since the hydrophilic block is still water-soluble at this time (when heated to a temperature higher than the cloud point), the polymer of the present invention is a hydrophobic association part between blocks having a cloud point in water. A hydrogel having a three-dimensional network structure with a cross-linking point as a cross-linking point is generated. When the temperature of this hydrogel is cooled again to a temperature lower than the cloud point of the “block having cloud point” existing in the molecule, the block having the cloud point becomes water-soluble and the crosslinking point due to hydrophobic association is released. The hydrogel structure disappears and the polymer of the present invention becomes a complete aqueous solution again. Thus, the sol-gel transition of the polymer of the present invention in a preferred embodiment is based on reversible changes in hydrophilicity and hydrophobicity at the cloud point of the block having the cloud point present in the molecule. Therefore, it has complete reversibility in response to temperature changes.
(Gel solubility)
As described above, the hydrogel-forming polymer of the present invention containing at least a polymer having a sol-gel transition temperature in an aqueous solution is substantially water-insoluble at a temperature (h ° C.) higher than the sol-gel transition temperature. And reversibly water soluble at a temperature (i ° C.) lower than the sol-gel transition temperature.
The high temperature (h ° C.) is preferably a temperature that is 1 ° C. or more higher than the sol-gel transition temperature, and more preferably a temperature that is 2 ° C. or more (especially 5 ° C. or more) higher. The term “substantially water-insoluble” means that the amount of the polymer dissolved in 100 mL of water at the temperature (h ° C.) is 5.0 g or less (more preferably 0.5 g or less, particularly 0.1 g or less. ) Is preferable.
On the other hand, the above-mentioned low temperature (i ° C.) is preferably 1 ° C. or more lower than the sol-gel transition temperature (in absolute value), more preferably 2 ° C. or higher (particularly 5 ° C. or higher). preferable. The “water-soluble” means that the amount of the polymer dissolved in 100 mL of water at the temperature (i ° C.) is 0.5 g or more (more preferably 1.0 g or more). Furthermore, “reversibly water-soluble” means that the aqueous solution of the hydrogel-forming polymer is once gelled (at a temperature higher than the sol-gel transition temperature), even after the gelation. At a lower temperature, it means to exhibit water solubility as described above.
The polymer preferably has a 10% aqueous solution at 5 ° C. and a viscosity of 10 to 3,000 centipoise (more preferably 50 to 1,000 centipoise). Such viscosity is preferably measured under the following measurement conditions, for example.
Viscometer: Stress-controlled rheometer (Model name: CSL 500, manufactured by Carrie Med, USA)
Rotor diameter: 60mm
Rotor shape: parallel plate
Measurement frequency: 1 Hz (Hertz)
The aqueous gel solution of the hydrogel-forming polymer of the present invention is substantially not dissolved even if it is gelled at a temperature higher than the sol-gel transition temperature and then immersed in a large amount of water. The characteristics of the floating carrier can be confirmed, for example, as follows.
That is, 0.15 g of the hydrogel-forming polymer of the present invention is dissolved in 1.35 g of distilled water at a temperature lower than the sol-gel transition temperature (for example, under ice cooling) to prepare a 10 W% aqueous solution. The aqueous solution was poured into a plastic petri dish having a diameter of 35 mm and heated to 37 ° C. to form a gel having a thickness of about 1.5 mm in the petri dish, and then the weight of the whole petri dish containing the gel (J-gram) is measured. Next, after the whole petri dish containing the gel was allowed to stand at 37 ° C. for 10 hours in water in 250 mL, the weight (k grams) of the whole petri dish containing the gel was measured, and the dissolution of the gel from the gel surface was measured. Evaluate presence or absence. At this time, in the hydrogel-forming polymer of the present invention, the weight reduction rate of the gel, that is, (j−k) / j is preferably 5.0% or less, more preferably 1.0%. Or less (particularly 0.1% or less).
The aqueous solution of the hydrogel-forming polymer of the present invention is gelled at a temperature higher than the sol-gel transition temperature, and then immersed in a large amount of water (by volume, about 0.1 to 100 times the gel). Even so, the gel does not dissolve over a long period of time. Such a property of the polymer used in the present invention is achieved by, for example, the presence of two or more (plural) blocks having a cloud point in the polymer.
On the other hand, when a similar gel was prepared using the aforementioned Pluronic F-127 in which polyethylene oxide was bonded to both ends of polypropylene oxide, the gel was completely dissolved in water after standing for several hours. The inventors have found that this is the case.
From the standpoint of suppressing the non-gelling cytotoxicity to the lowest possible level, the concentration in water, that is, {(polymer) / (polymer + water)} × 100 (%), 20% or less (and 15 It is preferable to use a hydrogel-forming polymer that can be gelled at a concentration of not more than%, particularly not more than 10%.
(Liquid component)
The liquid component (or dispersion medium) that can be used in constructing the carrier (gel-forming composition) of the present invention together with the above-described hydrogel-forming polymer has gel-forming properties as described above. There is no particular limitation as long as the floating carrier exhibits a thermoreversible sol-gel transition in which it is in a sol state at a low temperature and a gel state at a high temperature, and the gel-forming floating carrier is substantially insoluble in a high temperature gel state. This liquid component exhibits a substantially liquid state at a temperature at which the carrier of the present invention should be used in a sol state.
As such a liquid component, various inorganic liquids, organic liquids, and combinations or mixtures of two or more thereof can be used. In an embodiment in which the carrier of the present invention should be used as a component derived from a living body, this liquid component is preferably a hydrophilic or water-soluble liquid, and further contains a water-containing liquid (particularly, containing 80% by mass or more of water). Liquid).
(Other ingredients)
The floating carrier of the present invention contains at least the polymer having the above-mentioned sol-gel transition temperature, but may contain other components as necessary. Examples of “other components” in such an embodiment include antibiotics, anticancer agents, ECM such as collagen, local chemical mediators described later, hormones such as insulin and cell growth factor, foreign genes, and the like.
(Floating substances)
In the present invention, the floating substance to be suspended by the above-described floating carrier is not particularly limited. That is, the suspended substance may be derived from a living body or not. Preferred embodiments of the suspended substance are as follows.
Specific gravity: In the carrier of the present invention in a sol state, it can be substantially separated from the carrier by centrifugation (for example, conditions of 500 to 10,000 rpm, 100 to 10,000 G, about 5 to 30 minutes). Have specific gravity.
Examples of the substance to be suspended include the following.
Cells, colloidal particles (hydrophilic and / or hydrophobic), liquids, gases.
(Proliferation of fibroblasts)
In an embodiment in which the carrier of the present invention should be used as a biological component for the purpose of growing undifferentiated cells in an undifferentiated state, in the hydrogel formed by the hydrogel-forming polymer constituting the carrier, Preferably, fibroblasts do not substantially grow in the gel. Fibroblasts usually show significant proliferation with dendritic morphological changes characteristic of fibroblasts in monolayer culture on a cell culture dish (plate) or in a collagen gel. On the other hand, in the hydrogel of the present invention, fibroblasts do not substantially proliferate while maintaining the single cell form.
The proliferation of fibroblasts can be evaluated, for example, by the following method (see, Tsuyoshi Yoshikawa, Ken Tsukikawa, St. Marianna University School of Medicine, Vol. 28, No. 4, 161-170 (2000)). A normal human lung fibroblast is obtained by stirring and dissolving the hydrogel-forming polymer constituting the suspension carrier of the present invention in a culture solution such as RPMI 1640 (Life Technologies, NY, USA) at a low temperature (eg, 4 ° C.). (Normal Human Lung Fibroblasts, NHLF, manufactured by Takara Shuzo Co., Ltd.)) 6 × 10 ⁴ Disperse to a cell density of 1 cell / mL. 0.2 mL of this NHLF dispersion was added to a 24-well plate (material: plastic, the size of one well was 15 mm long, 15 mm wide, and about 20 mm deep; commercially available products such as Becton-Dickinson's trade name: After injecting into each well of Multiwell) and gelling at 37 ° C., 0.4 mL of the culture solution was added, and 37 ° C., 5% CO 2 was added. ₂ Incubate under atmospheric pressure. The state of fibroblast proliferation is confirmed by observation with a phase contrast microscope over time (for example, 0, 1, 3, 7 days).
(Proliferation rate of fibroblasts)
Furthermore, the proliferation rate of fibroblasts during the culture period can be measured by the following method utilizing the enzyme activity. After culturing fibroblasts in the floating carrier of the present invention for a predetermined period, the floating carrier is lowered to a temperature lower than its sol-gel transition temperature (for example, 10 ° C. lower than the sol-gel transition temperature). Then, 50 μL of WST-8 reagent (manufactured by Dojindo) is added to each well as a reagent for measuring succinate dehydrogenase activity. The 24-well plate is reacted at 37 ° C. for 10 hours and then stored at about 4 ° C. for 1 hour to make a completely uniform aqueous solution. The aqueous solution is dispensed into a 96-well plate by 200 μL, and the absorbance (OD (450)) is measured at 450 nm (reference wavelength: 620 nm) using a microplate colorimeter. It has been confirmed that this OD (450) and the number of living cells are in a proportional relationship (for example, the document Furukawa, T. et al, “High in vitro-in vitro correlation of drug response-suppressed-stressed-suppressed-the-supplemented-suppressed-the-supple- Dimensional history and MTT end point ”, Int. J. Cancer 51: 489, 1992). That is, the proliferation rate of fibroblasts is determined by the ratio between the absorbance at the start of culture (OD (450)) and the absorbance after culture (OD (450)).
In the present invention, the proliferation rate of fibroblasts after culturing at 37 ° C. for 3 days is in the range of 70% to 200%, further 80% to 150%, more preferably 90% to 120%. desirable.
(Chemical mediator)
For proliferation and differentiation of undifferentiated cells, not only cells such as progenitor cells but also various chemical mediators such as cell growth factors that promote differentiation and proliferation are usually required. These chemical mediators are normally secreted from the cells themselves. However, in order to promote the regeneration efficiently, it is effective to replenish the floating carrier of the present invention with these chemical mediators from the outside in advance.
The chemical mediators are: 1) a local chemical mediator that acts only in the immediate vicinity of the cell, 2) a neurotransmitter that is secreted from a neuron and has a very short effective action distance, and 3) Examples thereof include hormones that are secreted from endocrine cells and act on target cells throughout the body through blood flow and the like.
Examples of the local chemical mediator of 1) include proteins such as nerve cell growth factors, peptides such as chemotactic factors, amino acid derivatives such as histamine, and fatty acid derivatives such as prostaglandins.
Examples of the neurotransmitter 2) include low molecular weight substances such as amino acids such as glycine, low molecular weight peptides such as noradrenaline, acetylcholine, and enkephalin.
Examples of the hormone 3) include fibroblast growth factor (FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), liver VEGF, and VEGF. Cell growth factor such as hepatocyte growth factor (HGF), protein such as insulin, somatotropin, somatomedin, corticotropin (ACTH), parathyroid hormone (PTH), thyroid stimulating hormone (TSH), or glycoprotein, TSH Release factors, amino acid derivatives such as vasopressin, somatostatin, steroids such as cortisol, estradiol, testosterone, etc. It is.
(Floating / collecting method)
In the present invention, the above-mentioned floating carrier of the present invention is put into a sol state at a temperature lower than the sol-gel transition temperature, a suspended substance is added to the floating carrier, and the floating carrier is coated in a gel state at a temperature higher than the sol-gel transition temperature. The suspended matter can be retained, and then the suspended matter after the retention can be recovered again in a sol state lower than the sol-gel transition temperature. In the case of this collection | recovery, a well-known separation means (for example, centrifugation) can be used as needed.
Conditions other than those described above can be modified as appropriate according to the properties, properties, etc. of the suspended matter, with reference to a method for floating / recovering biological components described later.
(Floating / collecting method of biological components)
In order to seed and mix stem cells, progenitor cells or tissues containing them in the suspension carrier of the present invention, a hydrogel-forming polymer constituting the suspension carrier of the present invention is added to a culture solution such as RPMI 1640 (Life Technologies, NY, USA) with stirring and dissolution at a low temperature (for example, 4 ° C.), the suspended carrier of the present invention is added to and suspended in the state of an aqueous solution (sol) below its sol-gel transition temperature. You can do it. There is no particular limitation on the culture medium used here, and a target stem cell or progenitor cell that is easily proliferated and differentiated may be appropriately selected and used. Moreover, it is effective to contain the aforementioned chemical mediator that promotes the proliferation and differentiation of the target stem cells and progenitor cells in this culture solution.
In order to culture cells in the suspension carrier of the present invention, the suspension is heated to a temperature equal to or higher than the sol-gel transition temperature of the suspension carrier of the present invention (room temperature or 37 ° C.), and then gelled. Stem cells, progenitor cells or tissues containing them are cultured at the temperature (room temperature or 37 ° C.).
In the floating carrier of the present invention, the hydrogel-forming gel-forming composition is substantially water-insoluble in a high temperature gel state (culture temperature), so that a liquid medium is overlaid on the floating carrier of the present invention. The cells can be cultured by suspending the floating carrier of the present invention in a liquid medium. When undifferentiated cells proliferate, a large amount of nutrients are required, but the floating carrier of the present invention can supply the necessary nutrients from an external liquid medium. In addition, substances that inhibit cell growth, such as waste products generated by cells, can be discharged into an external liquid medium. As a result, the floating carrier of the present invention can promote cell growth as compared with conventional cell culture methods.
In order to recover the target tissue / organ in the floating carrier of the present invention and recover them from the floating carrier of the present invention, the floating carrier of the present invention containing the target tissue / organ is below the sol-gel transition temperature. After cooling to a temperature (for example, 4 ° C.), the floating carrier of the present invention is returned to the sol state, and the target tissue / organ and the floating carrier of the present invention are separated by a method such as centrifugation. In addition, since the floating carrier of the present invention can be easily diluted with water in a sol state lower than its sol-gel transition temperature, the fluidity can be further increased, and the cultured cells and organs can be easily recovered. Can do.
Since the suspension carrier of the present invention has the characteristics of suppressing the proliferation of fibroblasts and promoting the proliferation and differentiation of stem cells and progenitor cells, the target tissue / organ can be efficiently formed in the suspension carrier of the present invention.
(Recovered ES cells and / or embryoid bodies)
By using the above-described floating carrier of the present invention (a mode applied to a biological substance), it is easy to produce animal and plant cells (for example, protoplasts, ES cells, EBs, etc.) that need to be cultured in a floating state without adhering to the container wall. In addition to seeding and mixing, it can be suitably recovered. According to the present invention, it is possible to culture the animal and plant cells in a floating state without adhering to the vessel wall during the culture, and the medium can be changed during the culture. This is because the collected cells or cell mass can be easily recovered.
Accordingly, by using the floating carrier of the present invention, for example, ES cells and / or embryoid bodies can be suitably cultured and recovered.
In clinical application of ES cells, it is a precondition for use to culture in large quantities while maintaining undifferentiation. Regarding mouse ES cells, it has become clear that the use of LIF (leukemia inhibitory factor) can maintain undifferentiation, but the mechanism of maintaining undifferentiation has not been elucidated in primate ES cells including humans. (See Experimental Medicine, Vol. 21, No. 8 (Special Issue), “Cutting-edge of Stem Cell Research”, edited by Eiyuki Okano and Norio Nakajo, 2003, published by Yodosha).
Currently, it is reported that primate ES cells can be maintained undifferentiated by using mouse fibroblasts as a feeder (Nakatsuji N, Suemiri H, Embryonic stem cell lines of nonhuman primates, Scientific ref. Jun 26; 2 (6): 1762 (2002)). According to this report, when cynomolgus monkey ES cells are grown in a liquid culture medium that does not contain LIF, and in a flat culture using mouse fibroblasts as a feeder, one island-like colony is formed and the undifferentiated state is maintained. Has been. However, such co-culture with other animal cells (fibroblasts) is a serious problem in clinical application to humans.
On the other hand, by using the floating carrier of the present invention, mouse ES cells, cynomolgus monkeys, humans and other primate ES cells can be cultured in an undifferentiated state without using LIF and feeder cells. Thus, it is extremely advantageous in clinical application of ES cells that no co-culture with cells of other species is required. In addition, since the fibroblasts do not proliferate in the floating carrier of the present invention, it is possible to selectively proliferate only ES cells even if fibroblasts of other animals are mixed. It becomes.
Thus, the ES cells or embryoid bodies cultured and recovered using the floating carrier of the present invention have the following suitable characteristics.
<Characteristics of recovered ES cells>
Normally, ES cells proliferate while maintaining an undifferentiated state in the presence of LIF, but in the absence of LIF, they differentiate into mature cells such as nerves, blood cells, and muscles. However, ES cells cultured and collected using the suspension carrier of the present invention have a characteristic that they can maintain an undifferentiated state even in culture in the absence of LIF.
Maintenance of the undifferentiated state can be confirmed by analyzing the gene expression state with a DNA chip (microarray). If the gene expression of the cells after the culture does not change or the change is small compared to the gene expression of the ES cells before the culture, it is determined that the undifferentiated state is maintained. In addition, if the gene expression of the ES cell aggregate after culturing does not change compared to the gene expression of the ES cell before culturing, or the change is small, there is no or little contamination of cells differentiated into other cells. It is judged.
The maintenance of an undifferentiated state can also be confirmed from the formation of almond-shaped colonies when the recovered ES cells are cultured on feeder cells. The maintenance of the undifferentiated state can also be confirmed by creating a knockout mouse (chimeric mouse).
It can also be confirmed by observing the activity of alkaline phosphatase that the ES cells are maintained in an undifferentiated state. If the alkaline phosphatase activity is high, the cells are stained red when stained with Vector Red Alkaline Phosphatase Substrate Kit I (VECTOR).
Regarding the above-mentioned characteristic confirmation method, the document “Regenerative Medicine-From Basics of Tissue Engineering to Cutting-Edge Technology” Director: Norio Ohno, Masao Aizawa, Publisher: Takashi Yoshida, Publisher: NTS Corporation, Reference can be made to 2002, (Tokyo).
Since the recovered ES cells in the present invention maintain an undifferentiated state, they can be further used as ES cells (universal cells). Since the recovered ES cells are maintained in an undifferentiated state, differentiation can be induced into target cells and organs using various chemical mediators. The floating carrier of the present invention can also be used in an EB differentiation induction process using such a chemical mediator.
<Characteristics of recovered embryoid body (EB)>
EB cultured and recovered using the floating carrier of the present invention has a characteristic that it can maintain an undifferentiated state. It can confirm that EB is maintaining the undifferentiated state by the method similar to the case of said ES cell. The method for confirming that EB is still in an undifferentiated state can be found in the literature "Regenerative Medicine-From the Basics of Tissue Engineering to the Cutting-Edge Technology-" Director: Noriya Ohno, Masuo Aizawa, Publisher: Takashi Yoshida, Publisher: NTS Corporation, 2002, (Tokyo) can also be referred to.
Since the EB collected in the present invention maintains an undifferentiated state, it can be induced to differentiate into a target cell or organ using various chemical mediators. The floating carrier of the present invention can also be used for such EB differentiation induction process.
When the above-described hanging culture method is used to create EBs, EBs are not always formed from a single ES because separate ES cells gather at the bottom of the droplets to form EBs. . On the other hand, EBs cultured and recovered using the floating carrier of the present invention can form pure EBs because single (one) cells can proliferate to form EBs. Thus, the purity of EB can be confirmed by gene expression analysis as described above.
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is limited by a claim, and is not limited by a following example.

Production Example 1
10 g of a polypropylene oxide-polyethylene oxide copolymer (propylene oxide / ethylene oxide average polymerization degree of about 60/180, manufactured by Asahi Denka Kogyo Co., Ltd .: Pluronic F-127) was dissolved in 30 mL of dry chloroform, and in the presence of phosphorus pentoxide, Hexamethylene diisocyanate (0.13 g) was added, and the reaction was allowed to proceed for 6 hours under reflux at the boiling point. After distilling off the solvent under reduced pressure, the residue was dissolved in distilled water, and ultrafiltration was performed using an ultrafiltration membrane (Amicon PM-30) with a molecular weight cut off of 30,000 to obtain a high molecular weight polymer and a low molecular weight polymer. Fractionated. The obtained aqueous solution was frozen to obtain F-127 high polymer and F-127 low polymer.
The F-127 high polymer (hydrogel-forming polymer of the present invention, TGP-1) obtained as described above was dissolved in distilled water at a concentration of 8% by mass under ice cooling. When this aqueous solution was gently heated, the viscosity gradually increased from 21 ° C. and solidified at about 27 ° C. to form a hydrogel. When this hydrogel was cooled, it returned to an aqueous solution at 21 ° C. This change was repeatedly observed reversibly. On the other hand, the F-127 low polymer dissolved in distilled water at a concentration of 8% by mass below freezing did not gel at all even when heated to 60 ° C. or higher.
Production Example 2
160 mol of ethylene oxide was added by cationic polymerization to 1 mol of trimethylolpropane to obtain a polyethylene oxide triol having an average molecular weight of about 7000.
After dissolving 100 g of the polyethylene oxide triol obtained above in 1000 mL of distilled water, 12 g of potassium permanganate was gradually added at room temperature, and the reaction was allowed to proceed for about 1 hour. After removing the solid matter by filtration, the product was extracted with chloroform, and the solvent (chloroform) was distilled off under reduced pressure to obtain 90 g of a polyethylene oxide tricarboxylate.
10 g of the polyethylene oxide tricarboxylate obtained above and 10 g of polypropylene oxide diamino (propylene oxide average polymerization degree: about 65, manufactured by Jefferson Chemical Co., USA, trade name: Jeffamine D-4000, cloud point: about 9 ° C.) It melt | dissolved in 1000 mL of carbon tetrachloride, and after adding 1.2 g of dicyclohexylcarbodiimide, it was made to react under boiling point recirculation | reflux for 6 hours. After cooling the reaction solution and removing solids by filtration, the solvent (carbon tetrachloride) is distilled off under reduced pressure, and the residue is vacuum-dried, and the hydrogel of the present invention in which a plurality of polypropylene oxides and polyethylene oxides are combined. A forming polymer (TGP-2) was obtained. This was dissolved in distilled water at a concentration of 10% by mass under ice-cooling, and its sol-gel transition temperature was measured and found to be about 16 ° C.
Production Example 3
96 g of N-isopropylacrylamide (manufactured by Eastman Kodak), 17 g of N-acryloxysuccinimide (manufactured by Kokusan Chemical Co., Ltd.), and 7 g of n-butyl methacrylate (manufactured by Kanto Chemical Co., Ltd.) are dissolved in 4000 mL of chloroform, and nitrogen is added. After the substitution, 1.5 g of N, N′-azobisisobutyronitrile was added and polymerized at 60 ° C. for 6 hours. After the reaction solution was concentrated, it was reprecipitated (reprecipitated) in diethyl ether. The solid was collected by filtration and then vacuum dried to obtain 78 g of poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate).
To the poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate) obtained above, an excess of isopropylamine was added to obtain poly (N-isopropylacrylamide-co-n-butyl methacrylate). Obtained. The cloud point of this aqueous solution of poly (N-isopropylacrylamide-co-n-butyl methacrylate) was 19 ° C.
Chloroform 10 g of the above poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-n-butyl methacrylate) and 5 g of both ends aminated polyethylene oxide (molecular weight 6,000, manufactured by Kawaken Fine Chemical Co., Ltd.) It melt | dissolved in 1000 mL and made it react at 50 degreeC for 3 hours. After cooling to room temperature, 1 g of isopropylamine was added and allowed to stand for 1 hour, and then the reaction solution was concentrated, and the residue was precipitated in diethyl ether. The solid matter was collected by filtration and then vacuum-dried to form the hydrogel-forming polymer (TGP-3) of the present invention in which a plurality of poly (N-isopropylacrylamide-co-n-butyl methacrylate) and polyethylene oxide were bonded. )
The TGP-3 thus obtained was dissolved in distilled water at a concentration of 10% by mass under ice cooling, and the sol-gel transition temperature was measured to be about 21 ° C.
Production Example 4
(Sterilization method)
2.0 g of the above-described hydrogel-forming polymer (TGP-3) of the present invention is put into an EOG (ethylene oxide gas) sterilization bag (trade name: hybrid sterilization bag, manufactured by Hogi Medical Co., Ltd.), and an EOG sterilizer ( The bag was filled with EOG with Easy Pack (manufactured by Inoue Seieido) and left at room temperature all day and night. Further, after standing at 40 ° C. for half a day, the EOG was removed from the bag and aerated. The bag was sterilized by placing it in a vacuum dryer (40 ° C.) and leaving it for half a day with occasional aeration.
It was separately confirmed that the sol-gel transition temperature of the polymer was not changed by this sterilization operation.
Production Example 5
After dissolving 37 g of N-isopropylacrylamide, 3 g of n-butyl methacrylate and 28 g of polyethylene oxide monoacrylate (molecular weight 4,000, manufactured by NOF Corporation: PME-4000) in 340 mL of benzene, 2,2 ′ -0.8 g of azobisisobutyronitrile was added and reacted at 60 ° C for 6 hours. The obtained reaction product was dissolved by adding 600 mL of chloroform, and the solution was added dropwise to 20 L (liter) of ether to cause precipitation. The obtained precipitate was recovered by filtration, and the precipitate was vacuum-dried at about 40 ° C. for 24 hours, and then dissolved again in 6 L of distilled water, and a hollow fiber ultrafiltration membrane (H1P100 manufactured by Amicon Co., Ltd.) having a molecular weight cut-off of 100,000. -43) and concentrated to 2 liters at 10 ° C. The concentrated solution was diluted by adding 4 l of distilled water, and the above dilution operation was repeated. The above dilution and ultrafiltration concentration operations were further repeated 5 times to remove those having a molecular weight of 100,000 or less. What was not filtered by this ultrafiltration (remaining in the ultrafiltration membrane) was recovered and lyophilized to obtain 60 g of the hydrogel-forming polymer (TGP-4) of the present invention having a molecular weight of 100,000 or more. Obtained.
1 g of the hydrogel-forming polymer (TGP-4) of the present invention obtained as described above was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, the sol-gel transition temperature was 25 ° C.
Production Example 6
When the hydrogel-forming polymer (TGP-3) of Production Example 3 of the present invention was dissolved in distilled water at a concentration of 10% by mass and measured at η at 37 ° C., it was 5.8 × 10 ⁵ Pa · sec. there were. On the other hand, after agar was dissolved in distilled water at a concentration of 2% by mass at 90 ° C. and gelled at 10 ° C. for 1 hour, η at 37 ° C. was measured. 10 ⁷ Pa · sec).
Production Example 7
(Evaluation of fibroblast proliferation)
After sterilizing the hydrogel-forming polymer (TGP-3) of the present invention produced in Production Example 3 by the method of Production Example 4, 20% fetal bovine serum so that the final concentration of the polymer is about 8%. (FCS; manufactured by Dainippon Pharmaceutical, trade name: Fetal Calf Serum) and antibiotics (Life Technologies, trade name: penicillin; final concentration 10,000 U / mL) in RPMI-1640 (manufactured by Life Technologies) Was dissolved under stirring at 4 ° C. for 24 hours. This operation was performed aseptically.
Normal human lung fibroblasts (Normal Human Lung Fibroblasts, NHLF, manufactured by Takara Shuzo Co., Ltd.)) on the above-described floating carrier of the present invention (TGP-3 / RPMI)) at a cell density of 6 × 10 ⁴ cells / mL. Dispersed. After 0.2 mL of this NHLF dispersion was dispensed into each well of a 24 well plate [FAL bottom multiwell tissue culture plate (FALCON, Becton Dickinson & Company)] and gelled at 37 ° C., 0.4 mL of the culture solution was added. After addition, the cells were cultured at 37 ° C. and 5% CO _{2 at} atmospheric pressure. A total of 8 24-well plates were prepared for the 0th, 1st, 3rd, and 7th days, each for microscopic observation and fibroblast proliferation rate measurement. Separately, for comparison, without using TGP-3, an NHLF dispersion was prepared in which the cell density was 6 × 10 ⁴ cells / mL without using TGP-3. Similarly, 8 24-well plates were prepared. Then, the same culture test was conducted.
As a result of observation with a phase contrast microscope on a daily basis (0, 1, 3, 7 days), in the culture of the comparative example, a dendritic growth characteristic of fibroblasts was observed after 1 day, and confluent after 7 days. On the other hand, in the suspension carrier of the present invention, the state of proliferation was not observed until 7 days after the fibroblasts remained in the form of single cells.
After elapse of the predetermined culture days, the floating carrier was dissolved by lowering the 24 well plate to 4 ° C., and then the WST-8 reagent (manufactured by Dojin Chemical Co., Ltd.) as a reagent for measuring succinate dehydrogenase activity in each well. 50 μL was added. The 24 well plate was reacted at 37 ° C. for 10 hours and then cooled to 4 ° C. to make a completely uniform aqueous solution. The aqueous solution was dispensed into a 96-well plate by 200 μL, and the absorbance (OD (450)) was measured at 450 nm (reference wavelength: 620 nm) using a microplate colorimeter. The proliferation rate of fibroblasts was determined by the ratio of the absorbance at the start of culture (day 0) (OD (450)) and the absorbance after culture (after 1, 3, 7 days) (OD (450)). In the floating carrier of the present invention, the proliferation rate of fibroblasts after 1, 3 and 7 days was 105%, 120% and 125%, respectively, whereas in the comparative example, the fibroblasts after 1, 3 and 7 days The growth rates were 170%, 370%, and 420%, respectively.

After sterilizing the hydrogel-forming polymer (TGP-3) of the present invention produced in Production Example 3 by the method of Production Example 4, 15% calf fetal serum so that the final concentration of the polymer is 9.1% , 450 mM monothioglycerol, 10 mg / L Insulin's Modified Dulbecco's Medium (IMDM, manufactured by GIBCO) medium supplemented with insulin.
The sol-gel transition temperature of this aqueous solution (the floating carrier of the present invention) was 20 ° C. The iron ball (diameter 4 mm) sedimentation rate V ₃₇ in this aqueous solution at 37 ° C. was 0.001 mm / min or less, and the iron ball (diameter 4 mm) sedimentation rate V ₁₀ in this aqueous solution at 10 ° C. was 500 mm / min or more. It was.
Mouse ES cells (129SV, manufactured by Dainippon Pharmaceutical Co., Ltd.) were seeded at a concentration of 5 × 10 ³ cells / mL on the suspension carrier of the present invention, and the culture dish for bacteria (diameter 100 mm, previously warmed to 37 ° C.) 2 mL was added dropwise to) to form islands. Gelation occurred by leaving it to stand for 30 minutes under culture conditions of 5% CO ₂ and 37 ° C. An EB was prepared by culturing for 6 days under a culture condition of 5% CO ₂ and 37 ° C. as it was with a lid (FIG. 3). In the floating carrier of the present invention, the positional relationship of the cells at the time of seeding was preserved, and EB induction from ES cells that were single cells could be observed.
After incubation, 20-30 mL of PBS was added and allowed to stand on ice for 3-5 minutes, and then gently diluted by hand to easily dilute the gel. EB could be recovered by centrifuging this diluted solution.

After sterilizing the hydrogel-forming polymer (TGP-3) of the present invention produced in Production Example 3 by the method of Production Example 4, 15% calf fetus so that the final concentration of the polymer is 9.1%. It was dissolved in Iscove's Modified Dulbecco's Medium (IMDM, manufactured by GIBCO) medium supplemented with serum, 450 mM monothioglycerol, 10 mg / L insulin.
Mouse ES cells (129SV, manufactured by Dainippon Pharmaceutical Co., Ltd.) were seeded at a concentration of 5 × 10 ³ cells / mL on the suspension carrier of the present invention, and the culture dish for bacteria (diameter 100 mm, previously warmed to 37 ° C.) 2 mL was added dropwise to) to form islands. Gelation occurred by leaving it to stand for 30 minutes under culture conditions of 5% CO ₂ and 37 ° C. Over the gel, 20 mL of 15% calf fetal serum-containing IMDM medium warmed to 37 ° C. was overlaid so as to cover the gel. After culturing for 3 days under a culture condition of 5% CO ₂ and 37 ° C. with a lid on, the layered IMDM medium was removed by suction while maintaining the atmosphere at 37 ° C., and 20 mL of 15% calf warmed to 37 ° C. IMDM medium containing fetal serum was overlaid again and replaced with fresh medium. The mixture was further covered for 3 days under a culture condition of 5% CO ₂ and 37 ° C. with the lid on (FIG. 4). The growth rate of EB was faster than that of Example 1 due to the effect of the overlay medium and the medium exchange.
The layered IMDM medium was removed by suction while maintaining the atmosphere at 37 ° C., 20-30 mL of PBS was added, and the mixture was allowed to stand on ice for 3-5 minutes. After gently shaking by hand, the gel could be easily diluted. EB could be recovered by centrifuging this diluted solution.
Comparative Example 1
Iscove's supplemented with commercially available methylcellulose-containing IMDM medium (ES-Cult ™, M3120, StemCell Technologies, methylcellulose concentration 2.5%) with 15% calf fetal serum, 450 mM monothioglycerol, 10 mg / L insulin It was diluted with Modified Dulbecco's Medium (IMDM, manufactured by GIBCO) medium to prepare a methylcellulose concentration of 1%. This floating carrier did not have a sol-gel transition temperature in the range of 0 ° C to 45 ° C. The 37 iron ball of the floating carrier in ° C. (diameter 4 mm) sedimentation rate _{V 37} is 500 mm / min or more (by settling too fast, the measurement difficult), and iron ball in the aqueous solution at 10 ° C. (diameter 4 mm) sedimentation rate _{V 10} also was 500mm / min or more.
Mouse suspension cells (129SV, manufactured by Dainippon Pharmaceutical Co., Ltd.) were seeded at a concentration of 5 × 10 ³ cells / mL on this suspension carrier (methylcellulose culture carrier) and pre-warmed to 37 ° C. for bacterial culture dishes ( EB was prepared by dropping 2 mL onto a 100 mm diameter) and culturing for 6 days under 5% CO ₂ and 37 ° C. culture conditions (FIG. 3). Since the methylcellulose culture carrier is a high-viscosity liquid, the EB that gradually grows settles at an early stage, adheres to the bottom of the culture dish, and the generation of ES cell-derived fibroblast-like adhesive cells was observed.
After culturing, 20-30 mL of PBS was added and allowed to stand on ice for 3-5 minutes, and then gently shaken by hand, but the methylcellulose culture carrier could not be easily diluted. Further, suction and discharge were repeated using a pipette. However, since the liquid medium was not overlaid, the drying of the methylcellulose medium proceeded, and dilution was not easy. In addition, contamination of ES cell-derived fibroblast-like adhesive cells could not be avoided in the collected EB.

As a medium for mouse ES cells, a medium with LIF and a medium without LIF were prepared in advance. As the medium with LIF, a prepared medium for ES cells (Dainippon Pharmaceutical Co., Ltd.) was used. As a medium without LIF, ES cell D-MEM solution (Dainippon Pharmaceutical Co., Ltd.), ES cell serum 15%, ES cell nucleoside solution (100 ×), ES cell non-essential amino acid solution (100 ×), 100 X Penicillin-Streptomycin-Glutamine, liquid (manufactured by GIBCO) 1% each and βME (manufactured by Sigma) 110 μM were used.
After sterilizing the hydrogel-forming polymer (TGP-3) of the present invention prepared in Production Example 3 by the method of Production Example 4, the medium with LIF was adjusted so that the final concentration of the polymer was 9.1%. Each of the LIF-free medium was dissolved at 4 ° C. to prepare the floating carrier of the present invention (with LIF) and the floating carrier of the present invention (without LIF).
Mouse ES cells (129SV, manufactured by Dainippon Pharmaceutical Co., Ltd.) were dispersed at 4 ° C. in each of the suspension carrier of the present invention (with LIF) and the suspension carrier of the present invention (without LIF) at 8 × 10 ³ cells / ml. . 500 μL of each dispersion was dispensed at 4 ° C. into each well of a 6-well plate and allowed to stand in a 37 ° C. incubator for 15 minutes to gel the floating carrier of the present invention. Each ES cell dispersion gel (with and without LIF) was overlaid with 4 ml of each ES cell medium (with and without LIF) heated to 37 ° C., and cultured for 5 days without medium change in a 37 ° C. incubator. did.
The overlay medium was removed, 4 ml of phosphate buffer (PBS, pH 7.4) was added, and the gel was dissolved by shaking for about 15 minutes while cooling in ice. After the gel was completely dissolved, the cell clumps (spheroids) in the wells were sedimented and collected, and about 80 μL containing the collected cell clumps were separately prepared in 4% paraformaldehyde-phosphate buffer (PFA, (4) (manufactured by WAKO) and fixed at room temperature for 10 minutes. The cell mass (spheroid) in the well was collected, and about 80 μL containing the collected cell mass was added to 4 ml of PBS in a separately prepared 6-well plate and washed.
The spheroids in the wells were collected (about 40 μL), transferred to an alkaline phosphatase staining solution (400 μL) in a 1.5 mL centrifuge tube, and allowed to stand at room temperature in a dark place for 35 minutes for staining. The alkaline phosphatase staining solution was prepared by adding 2 drops of Reagent 1 to 5 ml of 100 mM Tris-HCl buffer (Tris-HCl, pH 8.2) using Vector Red Alkaline Phosphatase Substrate Kit I (manufactured by VECTOR). Then, 2 drops of Reagent 2 liquid were added and stirred, and 2 drops of Reagent 3 liquid were further added and stirred well. After staining, centrifugation was performed at 8000 × g for 3 minutes, and the precipitated spheroid (about 120 μL) was redispersed in PBS (4 ml) and observed with a phase contrast microscope (magnification 100 times).
Both mouse ES cells cultured in a medium with LIF (FIG. 6) and mouse ES cells cultured in a medium without LIF (FIG. 7) were stained in red by alkaline phosphatase staining, and were cultured using the suspension carrier of the present invention. The cells were shown to have high alkaline phosphatase activity, ie, remain undifferentiated. In particular, the mouse ES cells cultured in the LIF-free medium shown in FIG. 7 were stained more strongly in red, suggesting that they were more undifferentiated.
Comparative Example 2
The bottom of the culture flask (FALCON, culture area 25 cm ² ) was familiarized with 2 ml of a 0.1% gelatin solution for ES cells (Dainippon Pharmaceutical), allowed to stand at room temperature for 4 hours, then washed twice with 2 ml of PBS and air-dried. Primary mouse embryonic fibroblasts (hygromycin resistant, treated with mitomycin C) (Dainippon Pharmaceutical Co., Ltd.) are seeded at 1.2 × 10 ⁶ per flask, and the medium is added to DMEM (GIBCO) containing 10% FBS (GIBCO). 100% Penicillin-Streptomycin-Glutamine, liquid (GIBCO) added at 1% was added and cultured for 1 day, which was used as feeder cells. The feeder cell medium was removed and replaced with a prepared medium for ES cells (Dainippon Pharmaceutical), and mouse ES cells (Dainippon Pharmaceutical) (129SV: passage number 15-30) were seeded at 1 × 10 ⁶ per flask. . After changing the medium every day and culturing for 3 days, the medium was removed and washed twice with 5 ml of PBS. 2 ml of 4% paraformaldehyde-phosphate buffer (PFA, manufactured by WAKO) was added and fixed at room temperature for 10 minutes. After washing twice with 2 ml of PBS, 2 ml of the same alkaline phosphatase staining solution as in Example 3 was added, and the mixture was allowed to stand in a dark place at room temperature for 35 minutes for staining. The plate was washed twice with 2 ml of PBS and then observed with a phase contrast microscope (magnification 100 times). Mouse ES cells cultured in the presence of LIF on feeder cells were stained with alkaline phosphatase, and as a result, they were stained pale red as shown in FIG. This shows that the undifferentiated nature of ES cells is lower than that in the suspension carrier of the present invention of Example 3 (without LIF), even though cultured on feeder cells.

After sterilizing the hydrogel-forming polymer (TGP-3) of the present invention prepared in Production Example 3 by the method of Production Example 4, the medium for cynomolgus monkey ES is such that the final concentration of the polymer is 9.1%. It was dissolved in a basic medium for cynomolgus monkey ES (ESBM-101, manufactured by Asahi Techno Glass) supplemented with a medium additive for ES cells (ESMS-201, manufactured by Asahi Techno Glass). After dispersing 1 × 10 ⁵ cynomolgus monkey ES cells (CMES-001, manufactured by Asahi Techno Glass) in 0.5 ml of the floating carrier of the present invention, and placing 0.5 ml of the floating carrier containing the cells in the center of a 6-well plate in a drop shape The mixture was kept at 37 ° C. in an incubator for 30 minutes, and after the floating carrier was gelled, 10 ml of the above medium for cynomolgus monkey ES was overlaid and three-dimensional culture was performed. The stratified medium was changed every other day, and cynomolgus monkey ES cells formed EB-like spherical cell masses in mouse ES cells that were completely different from flat culture by culturing for one week.
Further, this spherical cell mass was collected in the same manner as in Example 3, and stained with alkaline phosphatase to confirm undifferentiation, and observed with a phase contrast microscope (400 magnifications). As a result, the collected cynomolgus monkey ES cell mass was strongly stained red as shown in FIG. 9, and the cynomolgus monkey ES cells cultured using the suspension carrier of the present invention have high alkaline phosphatase activity, that is, undifferentiated. It was shown to be maintained. By using the floating carrier of the present invention, it was possible to culture primate ES cells while maintaining undifferentiation without using other animal fibroblasts (feeder cells).
Comparative Example 3
Mouse fetal fibroblasts (manufactured by Asahi Techno Glass) in feeder medium (feeder cell basic medium (DMEM, Asahi Techno Glass) supplemented with feeder cell medium additive (Asahi Techno Glass)) 1 × 10 ⁵ cells / ml Then, 5 ml per seed was inoculated with gelatin coated DISH (diameter 60 mm, Asahi Techno Glass) for ES cells. The cells were cultured for 1 day and used as feeder cells. The feeder cell medium was removed, and cynomolgus monkey ES cells (CMES-001, manufactured by Asahi Techno Glass Co., Ltd.) dispersed in the cynomolgus monkey ES medium were seeded. After culturing for 5 days without exchanging the medium, alkaline phosphatase staining was performed in the same manner as in Comparative Example 2 and observed with a phase contrast microscope (400 magnifications). As a result, as shown in FIG. This shows that the undifferentiated nature of cynomolgus monkey ES cells is lower than that of Example 4 despite culturing on feeder cells.

As described above, since the floating carrier of the present invention is composed of a hydrogel-forming polymer that gels at a low temperature and in a sol state at a low temperature, the suspended substance ( For example, undifferentiated stem cells, progenitor cells, or tissues containing them) can be seeded or dispersed, mixed, and the suspension carrier of the present invention is gelled at an appropriate treatment (for example, culture) temperature as it is. With the floating carrier in a gel state, the suspended matter (for example, undifferentiated stem cells) can be treated (for example, cultured) without being brought into contact with the container wall containing the floating carrier.
In the floating carrier of the present invention, since the hydrogel-forming gel-forming composition is substantially water-insoluble in a high temperature gel state (treatment temperature), a liquid component (for example, a liquid medium) is formed on the floating carrier of the present invention. ), And the suspended carrier of the present invention is suspended in a liquid component for treatment (for example, cell culture).
In an embodiment in which the carrier of the present invention is used as a biological component, a large amount of nutrients are required when undifferentiated cells grow. However, the floating carrier of the present invention replenishes the necessary nutrients from an external liquid medium. can do. In addition, substances that inhibit cell growth, such as waste products generated by cells, can be discharged into an external liquid medium. As a result, the floating carrier of the present invention can promote cell growth as compared with conventional cell culture methods.
In order to recover these from the floating carrier of the present invention after performing the desired treatment (for example, induction of tissues / organs) in the floating carrier of the present invention, the floating carrier of the present invention including the target suspended matter is collected. After cooling to a temperature below the sol-gel transition temperature (for example, 4 ° C.), the floating carrier of the present invention is returned to the sol state, and the target suspended matter and the floating carrier of the present invention are separated by a method such as centrifugation. It ’s fine. In addition, since the floating carrier of the present invention can be easily diluted with water in a sol state lower than its sol-gel transition temperature, the fluidity can be further increased and the suspended matter can be more easily recovered.
In an embodiment in which the carrier of the present invention is used as a component derived from a living body, the floating carrier of the present invention can suppress the growth of fibroblasts and can promote the proliferation and differentiation of stem cells and progenitor cells. The target tissue / organ can be efficiently formed in the carrier.

Claims (15)

A gel-forming floating carrier comprising at least a hydrogel-forming polymer; the floating carrier exhibits a thermoreversible sol-gel transition in which it is in a sol state at a low temperature and in a gel state at a high temperature; Substantially insoluble in water in the gel state,
The sedimentation rate of iron balls (diameter 4 mm) in the floating carrier is 1 mm / min or less at a temperature 16 ° C. higher than the sol-gel transition temperature and 5 mm / min at a temperature 6 ° C. lower than the sol-gel transition temperature. A floating carrier characterized by the above. The ratio (b / a) of the sedimentation rate (a) at a temperature 16 ° C higher than the sol-gel transition temperature to the sedimentation rate (b) at a temperature 6 ° C lower than the sol-gel transition temperature is 5 or more. 2. The floating carrier according to 1. A gel-forming floating carrier comprising at least a hydrogel-forming polymer; the floating carrier exhibits a thermoreversible sol-gel transition in which it is in a sol state at a low temperature and in a gel state at a high temperature; Substantially insoluble in water in the gel state,
A floating carrier characterized in that the sedimentation rate of iron balls (diameter 4 mm) in the floating carrier is 1 mm / min or less at 37 ° C and 5 mm / min or more at 10 ° C. The floating carrier according to claim 3, wherein a ratio (c / d) between a sedimentation rate (c) at 37 ° C and a sedimentation rate (d) at 10 ° C is 5 or more. The floating carrier according to any one of claims 1 to 4, wherein fibroblasts do not substantially grow in the floating carrier. The floating carrier according to any one of claims 1 to 5, wherein the hydrogel-forming polymer is a polymer in which a plurality of blocks having a cloud point and a hydrophilic block are combined. The floating carrier according to any one of claims 1 to 6, wherein the sol-gel transition temperature is higher than 0 ° C and not higher than 45 ° C. Furthermore, the floating support | carrier in any one of Claims 1-7 containing water. The floating carrier according to any one of claims 1 to 8, further comprising a chemical mediator. A gel-forming floating carrier comprising at least water and a hydrogel-forming polymer; wherein the floating carrier exhibits a thermoreversible sol-gel transition in which it is in a sol state at a low temperature and in a gel state at a high temperature; And substantially water insoluble in a high temperature gel state; the sedimentation rate of iron spheres (diameter 4 mm) in the suspended carrier is 1 mm / min or less at a temperature 16 ° C. higher than the sol-gel transition temperature; Using a floating carrier that is at least 5 mm / min at a temperature 6 ° C. below the sol-gel transition temperature;
The suspended carrier is made into a sol state lower than the sol-gel transition temperature, and a suspended matter is added to the suspended carrier,
Hold the suspended object in a gel state higher than the sol-gel transition temperature,
A floatation / recovery method characterized by recovering the suspended matter after being held again in a sol state lower than the sol-gel transition temperature. A gel-forming floating carrier comprising at least a hydrogel-forming polymer; the floating carrier exhibits a thermoreversible sol-gel transition in which it is in a sol state at a low temperature and in a gel state at a high temperature; A floating carrier that is substantially water-insoluble in a gel state and has a sedimentation rate of iron balls (diameter 4 mm) in the floating carrier of 1 mm / min or less at 37 ° C. and 5 mm / min or more at 10 ° C. Use;
The suspended carrier is made into a sol state lower than the sol-gel transition temperature, and a suspended matter is added to the suspended carrier,
Hold the suspended object in a gel state higher than the sol-gel transition temperature,
A floatation / recovery method characterized by recovering the suspended matter after being held again in a sol state lower than the sol-gel transition temperature. The suspension / recovery method according to claim 10 or 11, wherein the suspended matter is a cell and / or tissue derived from a living body, and the cell and / or tissue derived from the living body are cultured in a floating carrier. The suspension / recovery method according to claim 12, wherein the suspended matter is an ES cell. An ES cell cultured and recovered by the method according to claim 12. Embryoid body (EB) characterized by being cultured and recovered by the method according to claim 12.

Images