EP1467719A2

EP1467719A2 - Method and device for feeding living cells into a biological body fluid

Info

Publication number: EP1467719A2
Application number: EP03731689A
Authority: EP
Inventors: Augustinus Bader
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-01-26
Filing date: 2003-01-22
Publication date: 2004-10-20
Also published as: WO2003061622A3; US20050118274A1; AU2003236832A1; CN100506281C; CN1622801A; WO2003061622A2; DE10202982A1

Abstract

Disclosed is a method for feeding living cells into a body fluid, particularly into a blood stream, according to which one or several of the cells are combined into capsule units by means of an enveloping substance surrounding the cells. In order to prevent coagulation or rejection, coagulation-inhibiting agents or coagulation-preventing structures are placed in or on the enveloping substance.

Description

Method and device for introducing living cells into a biological body fluid

The invention relates to a method and a device for introducing living cells into a biological body fluid, in particular into a blood stream, wherein one or more of the cells are combined to form capsule units by a covering substance surrounding the cells.

The invention also relates to a device for carrying out the method and capsule units therefor.

It is known, for example, to connect the patient to a liver reactor in the event of liver failure. However, this requires a plasma separation that separates the cellular components of the blood, in particular the red blood cells, from the actual plasma, which still contains the fibrin. It is not possible for the blood to be passed directly through the liver reactor, in which liver cells are encapsulated, ie in which several cells are combined to form larger capsule units by means of an enveloping substance surrounding the cells, since the blood would clot in the liver reactor , For this reason, a plasma separator would have to be interposed, in which the plasma is separated from the blood. It would be disadvantageous, however, that the capillaries present in the plasma separator would clog relatively soon, so that this would become ineffective and accordingly high effort and costs would have to be exchanged. This would generally be the case after eight to ten hours. A much more serious disadvantage, however, would be that the plasma separation or blood separation would result in a hymolysis in the patient, which would be traumatic for the patient and could even lead to death if the treatment was prolonged. In previous bioreactors, the biological systems were either directly exposed to a plasma without protection and thus triggered coagulation and complement activating, or positioned behind a plasma-separating membrane. A serious disadvantage of the plasma separators used is that the so-called membrane fouling occurs, ie a blockage due to deposits on the pores. As a result, these systems have to be replaced at short notice.

It is known that a damaged liver is able to regenerate itself if there are still healthy liver cells. If one could connect a patient to a liver reactor for a longer period of time, a damaged liver, e.g. poisoning or tumor surgery, the ability to regenerate if given enough time. However, several days are required here, which leads to the problems mentioned above.

The present invention is therefore based on the object of providing a method and a device with which body fluid, for example blood, can be treated directly, and disadvantageous plasma separation can be dispensed with. According to the invention, this object is achieved by a method for introducing living cells into a body fluid, in each case a plurality of cells surrounded by an enveloping substance are combined to form capsule units, and in order to avoid coagulation or rejection reactions in or onto the enveloping substance, anticoagulant agents or structures which prevent coagulation are brought become.

A device for carrying out this method is provided with a container which is equipped with an inlet for body fluid, e.g. Blood, and an outlet for blood is provided, in which a plurality of capsule units are arranged in the container, each of which is formed by a plurality of cells surrounded by an enveloping substance, the enveloping substance being provided with anticoagulant or anticoagulant structures.

According to the invention, anticoagulants or anticoagulant structures are now added to the cells or the capsule units with the cells arranged therein. In this way e.g. a blood stream can be passed directly through the "bioreactor" created in this way, without separation through technical membranes, such as e.g. Capillaries in hollow fiber bioreactors. This means that no prior plasma separation is required with the resulting disadvantages.

According to the invention, the blood obtained directly from a patient can now be passed through the bioreactor without causing blood to clot. After the blood purification, the blood can then be returned to the patient directly. One of the main differences from the prior art is that this procedure can be extended over several days, which means that, for example, patients with liver damage are given the opportunity for the liver to regenerate again.

Another advantage of the method and the device according to the invention is that after cell extraction and formation of capsule units and a corresponding precultivation, the capsule units, e.g. are placed in a bag-like container as a bioreactor, can freeze. In order to . it is possible to keep such bioreactors in stock and to be able to use them on a patient immediately after thawing.

As a means for measures that can prevent blood clotting, e.g. active anticoagulants, such as heparins, hirudins or prostaglandins and thromboxane structures, are suitable.

Another solution to this is to incorporate passively acting anticoagulant structures into the capsule units or to attach them to them. Passive anticoagulant structures prevent, for example, the red blood cells in the blood circulation from recognizing the capsule units as foreign bodies and triggering blood coagulation. In practice, the red blood cells are faked with a natural cell membrane surface. Lipids such as phosphatidylcholine, for example, are suitable for this, which can alternate with proteins or with glycoproteins. Likewise, glycocalix structures that are synthetically produced be prepared or obtained preparatively from erythrocyte surfaces, be integrated into or on the surfaces of the hybrid capsules, matrix (fibrin, collagen, albumin). As a result, they form a pattern of hydrophilic and hydrophobic groups, which in particular prevent the adhesion of other cells and blood clotting. The biological components can also be replaced by synthetic components, such as hydrophilic and hydrophobic plastics.

As can be seen, the cells arranged in the capsule units are cultivated directly in the blood stream which is passed through the bioreactor. In addition to the advantages already mentioned, this also results in a very significant cost reduction. Oxygenation of the bioreactor is not necessary during in-vivo use, since oxygen is directly introduced into the system by passing the blood through the red blood cells.

In practice, a patient can be treated with the bioreactor according to the invention in the same way as e.g. if you have a blood transfusion or kidney wash.

Polymers can be used as coating substances for the formation of capsule units with several cells. Examples of suitable polymers here are hydrogels, such as alginates, collagens, chitins, albumins and gels. However, biological polymers, such as polylactide, can also be used in the same way. A preferred field of application of the bioreactor is the cultivation of liver cells and of kidney cells or other cells that are attached to the bloodstream. The method described above relates to an extracorporeal insert which comes into contact only temporarily with body fluids, for example blood. However, the method according to the invention can also be used for intracorporeal systems, which are usually intended to remain in the body for life or as long as possible and can be given the coating according to the invention to avoid stopping forces.

An example are bioartificial vessels, implants, e.g. Heart valves and venous valves. These are different technical or biological structures, the surfaces of which are populated with different cells. These include e.g. also fibroblasts, smooth muscle cells and endothelial cells. In cases where the endothelialization is incomplete (especially with short or incomplete colonization processes), the underlying matrix is easily exposed. For this purpose, the matrix can consist of collagens, fibrin or synthetic polymers.

In order to avoid an immediate triggering of coagulation, the same factors or components as in the microcapsules can also be integrated into such hybrid structures in or above the matrix.

Another conceivable area of application is, for example, the use for diabetic patients. Here it is conceivable to use a bioreactor with cells that produce insulin as an implant in a patient's body. Because of the anticoagulant or anticoagulant structures according to the invention, this bioreactor is then not regarded by the body as a foreign body and is accordingly removed. bump. With corresponding islet cells made from embryonic stem cells or from umbilical cord blood or bone marrow cells, which are inserted into the capsule units and then injected directly into the patient's bloodstream, small glucose sensors could be formed in this way at various points in the organism Release glucose levels according to insulin.

The bioreactors are e.g. Hollow fiber bioreactors, fixed bed reactors, stirred fermenters, micro career systems and flat membrane bioreactors are possible.

Instead of blood as body fluid, the method according to the invention can e.g. can also be used with plasma or cerebrospinal fluid.

An embodiment of the invention is described in principle below with reference to the drawing.

It shows:

1 shows a device according to the invention; and

2 shows a greatly enlarged illustration of a capsule unit.

Fig. 1 shows schematically a container 1, which may have a bag shape, for example, with an inlet 2 for blood to be cleaned and an outlet 3 for the cleaned blood. A plurality of capsule units 4 are arranged in the interior of the container 1, in each of which a plurality of cells 5, for example Liver cells, (eg about 4 to 10 cell layers) are arranged. The cells 5 are surrounded by an envelope substance 6 and in this way form, for example, a spherical capsule unit 4. The connection between the envelope structure and the cells 5 can be made, for example, by appropriate mixing. Anticoagulant agents or anticoagulant structures 7 are introduced into the mixture or subsequently. If anticoagulants such as heparin are used, the anticoagulant will be mixed with the coating substance 6, so that the anticoagulants are not only arranged on the surface but also inside. Due to the porous design of the capsule units 4 or the envelope substance 6, the blood also flows through the capsule itself.

When using anticoagulant structures 7, care will be taken to ensure that these structures are preferably arranged on the surface of the capsule unit 4 or in its outer region, so that the red blood cells do not identify the capsule unit 4 as a foreign body. As coagulation-avoiding structures 7, e.g. Lipids can be used.

So that the capsule units 4 are not washed out of the container 1 during the blood washing or cultivation of the cells 5, the outlet 3 must be made correspondingly small. Alternatively, it is also possible to arrange a filter device 8 in front of the exit. Another possibility for retaining capsule units 4 in the container 1 can be a "magnetic trap". For this purpose, the capsule units 4 are additionally provided with tiny magnetic parts or magnetizable materials 9, with which the capsule sel units 4 react to a magnetic separator 10 (see dashed line in FIG. 1) with a magnet which is arranged in the exit region of the container 1. Here too, the magnetizable materials 9 will preferably be arranged on the surface or in the outer region of the capsule units 4.

The capsule units 4 are then held back by the magnetic separating device 10 and can be returned to the entrance area of the container 1 via a line 11 (shown in broken lines). A separation of the capsule units 4 from the blood or in order to avoid that the capsule units 4 are entrained with the bloodstream is basically possible without major problems. This is because red blood cells have a size of approximately 7 to 10 μm, while the capsule units 4 will generally be formed in a size or a diameter of 50 to 100 μm or 200 μm.

Claims

Patent claims

1. A method for introducing living cells into a biological body fluid, in particular into a blood stream, wherein one or more cells (5) surrounded by an enveloping substance (6) form capsule units

(4) are summarized, and in order to avoid coagulation or rejection reactions in or on the coating substance (6) anticoagulant or anticoagulant structures (7) are brought.

2. The method according to claim 1, characterized ge z e i chnet that heparins are used as anticoagulant (7).

3. The method of claim 1, d a d u r c h g e k e n n z e i c h n e t that hirudins are used as anticoagulant (7).

4. The method according to claim 1, characterized ge indicates that prostaglandins are used as anticoagulant (7).

5. The method according to claim 1, characterized ge zei chnet that thromboxane structures are used as anticoagulant (7).

6. The method of claim 1, d a d u r c h g e k e n n z e i c h n e t that a polymer is used as the coating substance (6).

7. The method of claim 6, d a d u r c h g e k e n n z e i c h n e t that a plastic polymer is used.

8. The method according to claim 7, since th r ch g e ke nn z e i chn e t that polylactide, polyurethane, polyester, gels, hydrogels or silicone are used as the plastic polymer.

9. The method of claim 6, d a d u r c h g e k e n n z e i c h n e t that biological polymers are used.

10. The method according to claim 9, since the use of alginates, collagens or chitins are used as biological polymers.

11. The method according to claim 1, since it is known that lipids are used as anticoagulant structures (7) and are applied at least to the surfaces of the capsule units (4).

12. The method of claim 11, since you r ch ge ke nn zei ch ne t that as lipids, phosphatidylcholine or other cell membrane components such as glycocalyx structures or glycocalyx components can be used.

13. The method of claim 11 or 12, d a d u r c h g e k e n n z e i c h n e t that the lipids (7) are bound to proteins and / or glycolipids and / or glycoproteins.

14. The method of claim 1, d a d u r c h g e k e n n z e i c h n e t that liver cells are used as cells (5).

15. The method according to claim 1 and 14, characterized in that the liver cells (5) bound in capsule units (4) are arranged in containers (1) which are provided with an inlet (2) and an outlet (3), the container (1) connected to a patient's bloodstream.

16. Capsule unit, in which a multiplicity of cells (5) are arranged in an enveloping substance (6) surrounding the cells (5), the enveloping substance (6) with anticoagulant agents or anticoagulant structures

(7) is provided.

17. Capsule unit according to claim 16, characterized in that heparin is provided as an anticoagulant (7).

18. A capsule unit according to claim 16, that a hududin is provided as an anticoagulant (7).

19. Capsule unit according to claim 16, d a d u r c h g e k e n n z e i c h n e t that prostaglandin is provided as an anticoagulant (7).

20. Capsule unit according to claim 16, that a thromboxane structures are used as anticoagulant (7).

21. Capsule unit according to claim 16, because ge ke n n z e i chne t that polymers are provided as the coating substance (6).

22. Device for introducing living cells into a biological body fluid, in particular into a blood stream, with a container which is provided with an inlet (2) for the body fluid and with an outlet (3), one in the container (1) A large number of capsule units (4) are arranged, each of which is formed by a plurality of cells (5) surrounded by an enveloping substance (6), the enveloping substance (6) being provided with anticoagulant or anticoagulant structures (7).

23. The device according to claim 22, characterized in that the container (1) is provided in the area of the outlet (3) with a retaining device (8, 10) for the capsule units (4).

24. The apparatus of claim 23, d a d u r c h g e k e n n z e i c h n e t that the retaining device (8) is designed as a filter device.

25. The apparatus of claim 23, d a d u r c h g e k e n n z e i c h n e t that magnetic parts or magnetizable materials (9) are embedded in the capsule units (4), and that the retaining device (8) is provided with a magnetic separator (10).