JP2008199897A - Substrate for cell culture and method for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new means for culturing cells three-dimensionally and sterically growing the cells. <P>SOLUTION: The substrate for cell culture is composed of a fibrous sheet comprising calcium phosphates such as hydroxyapatite. When the cell culture is carried out by using the substrate for the cell culture, the fibrous sheet and fiber branches thereof are initially used as a scaffold and the cells stick to the scaffold and grow. Then, the cells gradually and mutually adhere even in positions separated from the scaffold to thereby start three-dimensionally and sterically growing. Finally, cell aggregates are formed so as to cover the whole substrate for the cell culture. That is, when the substrate for cell culture is used, the cells can be grown even in a part where the cells do not come into contact with the scaffold by mutual adhesion of the cells. Thus, the cells can three-dimensionally and sterically be grown. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cell culture substrate comprising a fibrous sheet containing calcium phosphates (such as hydroxyapatite), a high-density culture instrument comprising the substrate, a cell culture method, and the like. The present invention also relates to long-term continuous culture, three-dimensional stereoscopic culture, and the like.

  Cells can be broadly divided into suspension cells and adherent cells. Suspended cells are cells that do not require a scaffold for growth and proliferation, and adherent cells are cells that require a scaffold for growth and proliferation. Most stem cells important in regenerative medicine are adherent cells, and they are cultured by attaching the cells to an artificially prepared scaffold.

  As scaffolds for adherent cells, for example, synthetic polymers such as polystyrene, biological proteins such as collagen, and the like are used. In addition, various improvements have been made to promote cell adhesion to a base material to be used as a scaffold, or to enhance protein adsorptivity to a base material to be used as a scaffold. In addition, from the viewpoint of clinical application, attempts have been made to use a bioabsorbable material such as PGA (polyglycolic acid) as a scaffold base material.

For example, the following Patent Documents 1 to 3 are disclosed as prior documents related to the present invention. Patent Document 1 describes a cell culture substrate made of a gel containing a hydrophilic component and fine particles (hydroxyapatite is also exemplified). Patent Document 2 describes a cell culture substrate comprising a honeycomb structure containing a biodegradable polymer. Patent Document 3 describes a bone regeneration base material in which a bioabsorbable organic porous material (nonwoven fabric is exemplified as a form) and an osteophilic inorganic material (hydroxyapatite is exemplified) are combined. ing.
JP 2005-27532 A JP 2001-157574 A JP-A-2005-52224

  Conventional cell culture, including high density culture, is basically a monolayer culture in that each cell must adhere to the scaffold. Therefore, it is difficult to grow the cells three-dimensionally.

  Therefore, a main object of the present invention is to provide a novel cell culture means that can be three-dimensionally and three-dimensionally grown.

  In this invention, the base material for cell culture which consists of a fibrous sheet containing calcium phosphates is provided.

When cell culture is carried out using this cell culture substrate, initially, the fibrous sheet and its fiber branch are used as a scaffold, and the cells grow while adhering to the scaffold. When cells adhere to each other, they start to grow three-dimensionally and three-dimensionally, and finally, a cell mass is formed so as to cover the whole cell culture substrate.
That is, when this cell culture substrate is used, the cells can be grown three-dimensionally and three-dimensionally because the cells can be proliferated even in a portion where the cells are not in contact with the scaffold.

  Note that cell culture using this substrate is applicable to high-density culture, continuous long-term culture, and the like.

  According to the present invention, cells can be grown three-dimensionally and three-dimensionally.

  Hereinafter, a preferred embodiment for carrying out the present invention will be described.

<About cell culture substrate>
The cell culture substrate according to the present invention comprises a fibrous sheet containing calcium phosphates.

The fibrous sheet is preferably a non-woven fabric, but is not limited thereto.
Unlike nonwoven fabrics that are regularly woven such as meshes, fiber branches are irregularly bonded to each other, and have an appropriate space, thickness, and elasticity. Therefore, when cells adhere to each other at a portion where the cells and the scaffold (nonwoven fabric) are not in contact with each other, it is assumed that the nonwoven fabric has a function of maintaining an appropriate distance between the cells and promotes adhesion between the cells.

The material (raw material) of the nonwoven fabric is not particularly limited as long as it can contain calcium phosphates, and a known material can be used. Nonwoven fabric materials (raw materials) such as natural fibers (cotton, hemp, wool, etc.), regenerated fibers (rayon, cupra), semi-synthetic fibers (acetate, promix), synthetic fibers (nylon, polyester, acrylic, vinylon) , Polyvinyl chloride, vinylidene, polyolefin, polyurethane, polyclar, fluorocarbon, novoloid, etc.), inorganic fiber (glass fiber, carbon fiber, alumina fiber, silicon carbide fiber, slag fiber, metal fiber, etc.) .
Moreover, you may form a nonwoven fabric with a well-known biodegradable material, for example, PGA (polyglycolic acid), PLGA (copolymer of polylactic acid and polyglycolic acid), etc.

A relatively high fiber density of the nonwoven fabric is preferable in that the cell density (culture efficiency) can be increased. For example, when the fiber density of the nonwoven fabric is converted to the weight per unit area of the fibrous sheet, at least one in the range of 0.1 to 0.3 g / 100 cm ² is suitable for efficient cell culture. .

Examples of the calcium phosphates include primary calcium phosphate, secondary calcium phosphate, tertiary calcium phosphate, various apatites (for example, hydroxyapatite, fluorapatite, hydrochloric acid apatite, carbonate apatite, etc.).
It is assumed that calcium phosphates promote cell adhesion to the nonwoven fabric at the start of culture.

  A known technique can be used for the method of incorporating calcium phosphates into the nonwoven fabric, and is not particularly limited. Examples of the method include a method of applying calcium phosphates to a nonwoven fabric and a method of using a binder (adhesive). In the case of culturing cells used for regenerative medicine or the like, or when producing collagen to be applied to the human body, a method without using a binder is more preferable from the viewpoint of maintaining health.

<About equipment for high-density culture>
As described above, by using the cell culture substrate according to the present invention, the cells can be three-dimensionally and three-dimensionally grown. Therefore, the present invention is applicable to high-density culture.

  The high-density culture instrument according to the present invention is formed by placing a cell culture substrate in a container generally used for culture, such as a flask or a petri dish. One cell culture substrate may be used, or two to three layers may be used in a stacked state. When two to three cell culture substrates are stacked, there is an advantage that the cell density (culture efficiency) can be increased. In addition, this high-density culture instrument may include a medium, a culture reagent, a reagent or member for fixing the base material, and the like in advance.

<About cell culture method>
By using the cell culture substrate or the high-density culture instrument, highly efficient cell culture can be performed.

  That is, for example, after a predetermined medium is put in a culture container, a cell culture substrate is added, and the substrate is made to conform to the medium. Then, the cells are encased therein and cell culture is performed. A well-known thing can be used for a culture medium.

The use of this cell culture method has the following advantages.
(1) This cell culture method is applicable not only to human fibroblasts but also to stem cells and most other adherent cells.
(2) By using this cell culturing method, three-dimensional and three-dimensional cell culture becomes possible, so that high-density culture can be performed without complex and three-dimensional scaffolding. In addition, cell density and culture efficiency can be increased.
(3) In this cell culture method, continuous culture can be performed for a long period of time while maintaining the proliferation of the cells. That is, continuous culture can be performed for a long period of time only by periodic medium exchange without performing subculture.
(4) In this cell culture method, cells that are not in contact with the scaffolds adhere to each other to proliferate three-dimensionally and three-dimensionally. That is, by performing continuous culture, the cells can proliferate even without a scaffold. Therefore, for example, when a fibrous sheet is formed of a biodegradable material, there is a possibility that a tissue / organ can be constructed using this cell culture method, and there is a possibility that it can be applied to regenerative medicine.

In addition, in this cell culture method, it is assumed that the reason why long-term continuous culture is possible is based on the following mechanism.
When cell culture is performed using the cell culture substrate according to the present invention, the cells adhere to each other at a portion where the cells and the scaffold are not in contact with each other, and a structure similar to the intercellular gap in the living body is constructed. Thereby, individual cells can directly metabolize using a medium to synthesize necessary substances themselves, thereby avoiding necrosis.

  In Example 1, cell culture was performed using a nonwoven fabric coated with hydroxyapatite as a scaffold.

After the culture medium was put into a 75 cm ² flask, a nonwoven fabric (9 × 8 cm, sterilized) coated with hydroxyapatite was placed. Then, after the medium was acclimated to the nonwoven fabric, 1 × 10 ⁶ human normal fibroblasts were infused and cultured.
The medium was composed of FBS (final concentration 5%), antibiotics, L-ascorbic acid phosphate magnesium salt 0.2 mM, and the medium was exchanged at weekly intervals.

The results are as follows.
At the beginning of the culture, cells adhered to both the nonwoven fabric and the bottom of the flask, and more adhered to the bottom of the flask than the nonwoven. This is presumably because the bottom of the flask has a larger scaffold area.
One week after the start of the culture, it was confirmed that the cells adhered to the non-woven fibers so as to be entangled and proliferated (see FIG. 1).
One month after the start of the culture, it was confirmed that cells adhered to each other even in a portion where there was no fiber as a scaffold, and spread and expanded in a film shape (see FIG. 2).
Two months after the start of the culture, the cells further expanded in a film shape and proliferated, and the cells expanded three-dimensionally over the entire thickness of the nonwoven fabric (see FIG. 3).
Three months after the start of the culture, the cells spread in a film shape and covered the whole nonwoven fabric to form a three-dimensional cell mass (see FIG. 4).

  As described above, when a non-woven fabric coated with hydroxyapatite is used as a scaffold, cells adhere to each other and spread into a film even in a place where the cells and the scaffold (nonwoven fabric) are not in contact at all. It has been found that it can grow three-dimensionally.

  In Example 2, the number of adherent cells and the amount of collagen produced when a nonwoven fabric coated with hydroxyapatite was used were examined.

Using a nonwoven fabric coated with hydroxyapatite, culture was performed in the same manner as in Example 1, and the number of adherent cells per surface area in a 100% confluent state and the amount of collagen in the culture supernatant were measured. The number of adherent cells was counted after the cells were suspended by trypsin treatment. In addition, since the cells adhering to the nonwoven fabric were firmly bound, trypsin treatment was repeated three times at 37 ° C. for 1 hour.
Further, as a control, normal flask culture was performed using a 75 cm ² flask, and similarly, the number of adherent cells per surface area in a 100% confluent state and the amount of collagen in the culture supernatant were measured.
In addition, the culture surface area at the time of using a nonwoven fabric is 72 cm < ² >, and the culture surface area at the time of performing flask culture is 75 cm < ² >.

The results are shown in Table 1. In addition, at the time of cell culture, not only the nonwoven fabric but also the cells adhere to the bottom of the flask and grow. Therefore, a value obtained by subtracting “flask culture” from “culture using nonwoven fabric” is estimated as a value based on cells grown on the nonwoven fabric.

As shown in Table 1, when culturing using a nonwoven fabric coated with hydroxyapatite, the number of adherent cells is 5.3 times and the amount of collagen is 4.0 times that of flask culture (monolayer culture). Increased to. This is presumed to be because the cultured cells can be suitably attached to the nonwoven fabric and can be three-dimensionally grown.
Therefore, the above experimental results show that the nonwoven fabric coated with hydroxyapatite is suitable for cell culture, and that this nonwoven fabric can be applied to high-density culture.

  In Example 3, in the same cell culture as in Example 1, the case where hydroxyapatite was applied to a nonwoven fabric was compared with the case where it was not applied.

A non-woven fabric coated with hydroxyapatite and a non-coated non-woven fabric were prepared and cultured in the same manner as in Example 1. In this experiment, 5 × 10 ⁵ human normal fibroblasts were infused, and the medium was changed every 4 to 5 days.

5 and 6 are photomicrographs showing the state of the cells after 2 weeks from the start of culture. FIG. 5 is a photomicrograph when culture is performed using a non-woven fabric coated with hydroxyapatite, and FIG. 6 is a photomicrograph when culture is performed using a non-woven fabric coated with hydroxyapatite.
As shown in FIGS. 5 and 6, when a non-woven fabric coated with hydroxyapatite was used, cell attachment was observed with many fibers of the non-woven fabric (FIG. 5), whereas a non-woven fabric coated with hydroxyapatite was used. In this case, cell attachment could be observed only with a part of the fibers of the nonwoven fabric (FIG. 6).

  Further, one month after the start of culture, the amount of collagen produced by the cells (the amount of collagen in the culture supernatant) was measured. As a result, when using a nonwoven fabric coated with hydroxyapatite, the amount of collagen produced was 5.6 μg / ml. On the other hand, when a non-woven fabric not coated with hydroxyapatite was used, the amount of collagen produced was 1.3 μg / ml (a value excluding the amount of collagen produced by cells attached to portions other than the non-woven fabric).

  The above results indicate that application of hydroxyapatite to the nonwoven fabric promotes cell adhesion and proliferation, and promotes production of substances such as collagen.

  In Example 4, the suitable fiber density of the nonwoven fabric was examined in the cell culture shown in Example 1 and the like.

The density of the fibers of the nonwoven fabric can be adjusted by the weight per unit area of the nonwoven fabric. Therefore, three types of non-woven fabrics (samples 1 to 3) having different densities (weight per unit area) were prepared, and cultured in the same manner as in Example 1 and the like. In this experiment, 4 × 10 ⁵ human normal fibroblasts were infused.
Then, one month after the start of culture, the amount of collagen in the culture supernatant was measured.
In addition, as a control, a similar experiment was performed using a non-woven fabric not coated with hydroxyapatite.

In addition, the weight per unit area (10 cm × 10 cm) of the nonwoven fabric used (samples 1 to 3) is as shown in Table 2.
In the table, the “nonwoven fabric + HAp” column indicates the weight per unit area of the nonwoven fabric coated with hydroxyapatite.
In the table, the column “HAp” indicates the weight of hydroxyapatite contained in the nonwoven fabric. The weight of hydroxyapatite was obtained by measuring the weight of the residue when the nonwoven fabric was burned.
In the table, the column of “nonwoven fabric only” indicates the weight per unit area of the nonwoven fabric. The weight per unit area of the nonwoven fabric was calculated by subtracting the value (B) in the “HAp” column from the value (A) in the “nonwoven fabric + HAp” column.

The results are shown in Table 3.

The results in Table 3 indicate that cell culture is good when the weight per unit area of the nonwoven fabric is at least in the range of 0.12 (0.117188) to 0.20 (0.198804) g / 100 cm ^2. What can be done, that is, the density of the fibers of the nonwoven fabric in that case, indicates that it is at least suitable for cell culture.
Further, the larger the density of fibers (weight per unit area) of the nonwoven fabric, the greater the amount of collagen in the culture supernatant. This is presumed to be because the nonwoven fabric having a higher density has a larger number of cells that can be attached.
In Table 3, the control value is a value excluding the amount of collagen produced by cells adhering to portions other than the nonwoven fabric, as in Example 3.

  In Example 5, cell culture was performed using a plurality of nonwoven fabrics coated with hydroxyapatite.

After the culture medium was put into a 75 cm ² flask, a plurality of non-woven fabrics coated with hydroxyapatite were put in layers and cultured in the same manner as in Example 1 and the like. In this experiment, as in Example 3, 5 × 10 ⁵ human normal fibroblasts were infused, and medium exchange was performed at intervals of 4 to 5 days.
Then, after 3 months from the start of culture, the amount of collagen in the culture supernatant was measured.

The results are shown in Table 4.

  As shown in Table 4, when a plurality of non-woven fabrics coated with hydroxyapatite were stacked in layers, collagen production increased at least up to three. From this, it was found that a culture environment in which two or three non-woven fabrics coated with hydroxyapatite are suitable for adhesion and proliferation of cultured cells.

  In Example 6, in the same cell culture as in Example 1 and the like, the case where the nonwoven fabric was fixed and the case where the nonwoven fabric was not fixed were compared.

  In this experiment, Meviol Gel (registered trademark, manufactured by Meviol Co., Ltd., hereinafter the same) was used as a means for fixing the nonwoven fabric. Meviol gel is in a fluid sol form at low temperature conditions and a gel form at conditions above the transition temperature (for example, culture conditions). After mixing under low temperature conditions, the nonwoven fabric is fixed by transferring to culture conditions. it can.

A non-woven fabric (3 cm × 7 cm, sterilized) coated with a medium, hydroxyapatite, and meviol gel were placed in a 10 cm petri dish, and the non-woven fabric was fixed, followed by culturing in the same manner as in Example 1 and the like. In this experiment, 2 × 10 ⁵ human normal fibroblasts were infused, and the medium was changed every 4 to 5 days.
Then, two months after the start of culture, the amount of collagen in the culture supernatant was measured.
As a control, culturing was carried out in the same manner without adding mebiol gel.

The results are shown in Table 5.

  As shown in Table 5, the amount of collagen was greater when the nonwoven fabric was fixed. This is because when the non-woven fabric is not fixed, the non-woven fabric floats and moves to physically damage the cells attached to the bottom of the petri dish, killing the cells, and living due to the influence of the dead cells. This is presumed because the amount of collagen produced by the cells also decreases.

  In Example 7, in the same cell culture as in Example 1 and the like, a method of instilling cells after attaching the cells to microcarriers was examined.

First, cells were attached to the microcarrier. 2 × 10 ⁶ human fibroblasts were placed in a 10 cm petri dish, and 0.5 to 2.0 g of a microcarrier “CELLYARD beads (manufactured by PENTAX)” was added the next day and cultured for 1 to 3 days. Thereby, more than half of the cells adhered to the microcarrier.
Next, the microcarriers were discharged onto the nonwoven fabric placed in the flask and cultured in the same manner as in Example 1 and the like.

  As a result, in Example 1, it took about 2 months for the collagen production to reach 20 μg / ml or more, whereas in this experiment, the collagen production became the same or more in about 3 weeks.

  This result shows that when cells are sprinkled into a non-woven fabric coated with hydroxyapatite, it is better to swallow cells after attaching them to microcarriers than to swallow cells after trypsin treatment as usual. It shows that the establishment and growth of can be accelerated.

  In Example 8, when cell culture was performed using a nonwoven fabric coated with hydroxyapatite, it was examined what kind of cells the nonwoven fabric can be applied.

A culture medium was placed in a 25 cm ² flask, and then a non-woven fabric (3 × 6 cm, sterilized) coated with hydroxyapatite was placed. Various cells were inoculated and cultured in the same manner as in Example 1 and the like. The culture was continued for 3 months or more.
The various cells soaked in are listed in the left column of Table 6 below.

As a result, as shown in Table 6, it was possible to culture in all the cells that were tested. In addition, as long as the cell morphology and growth rate were observed, the effects of environmental changes such as medium changes were hardly observed.

  The above results show that the culture method according to the present invention can be applied to any cell.

  The present invention is applicable to the culture of almost all adherent cells. In addition, there is an advantage that long-term continuous culture is possible.

A photomicrograph (drawing substitute photo) one week after the start of culture. A photomicrograph (drawing substitute photo) one month after the start of culture. Photomicrograph (drawing substitute photo) 2 months after the start of culture. Micrograph (drawing substitute photo) 3 months after the start of culture. Photomicrograph (drawing substitute photo) when hydroxyapatite is applied. Photomicrograph (drawing substitute photo) when hydroxyapatite is not applied.

Claims (7)

  A cell culture substrate comprising a fibrous sheet containing calcium phosphates.   The cell culture substrate according to claim 1, wherein the fibrous sheet is a non-woven fabric.   The substrate for cell culture according to claim 1 or 2, wherein the calcium phosphates are hydroxyapatite.   The cell culture substrate according to any one of claims 1 to 3, which is used for three-dimensional three-dimensional culture. The weight per unit area of the nonwoven fabric, the cell culture substrate according to one of the preceding claims 2, characterized in that the 0.12~0.20g / 100cm ^2.   A high-density culture instrument comprising at least the cell culture substrate according to any one of claims 1 to 5. A cell culture method using the cell culture substrate according to any one of claims 1 to 5 or the high-density culture instrument according to claim 6.