WO1999021962A1

WO1999021962A1 - Modified microcarriers and their use in cell cryoconservation

Info

Publication number: WO1999021962A1
Application number: PCT/EP1998/006713
Authority: WO
Inventors: Nikolaos Dimoudis; Ingo Heschel; Julia Pasch; Günter RAU
Original assignee: Roche Diagnostics Gmbh
Priority date: 1997-10-25
Filing date: 1998-10-22
Publication date: 1999-05-06
Also published as: AU1335699A

Abstract

The invention relates to a method for immobilizing biological material, especially cells and unions of cells on microcarriers, characterized in that the volume of the microcarriers to be coated is irreversibly reduced by modifying the osmotic conditions surrounding the microcarriers and the pretreated microcarriers are subsequently coated with the biological material in the usual manner. According to the invention, a substantially improved adherence of the biological material on the microcarriers is achieved.

Description

Modified microcarriers and their use for cryopreservation of cells

The invention relates to improved microcarriers and their use for the cryopreservation of cells or cell assemblies.

The immobilization of tissue or cells on microcarriers is used to a large extent and advantageously in connection with the cultivation of cells in vitro and for the production of transplant material from autologous or allogeneic cells, in particular in dermatology.

Canadian patent CA-A 1215922 describes, for example, the microencapsulation of tissue or individual cells using a polymer which contains polylysine groups with a molecular weight between 10 and 30,000 Da. These microcapsules are immobilized on a biocompatible polymeric material that carries negatively charged groups. S.T. Boyce describes in Surgery 103 (1988) 421-431 the coupling of cultured human epidermal keratinocytes to an insoluble carrier consisting of collagen and chondroitin-6-sulfate. A skin transplant material is known from WO 89/03228, which consists of epidermal cells which are immobilized on a collagen carrier. EP-A 0 333 328 describes a method for skin grafting in which the cells to be transplanted are also immobilized on microcarriers. EP-A 0 242 305 preferably uses placental collagen type I and or type III carriers as such carriers. M.L. Williams describes in J. Cellular Physiology 136 (1988) 103-110 the cultivation of immobilized keratinocytes. WO 93/23088 describes biotherapeutically usable cell-coated microparticles in which epidermal cells are bound to conventional microcarriers. WO 96/12510 describes an improved method and a biomaterial which contains epithelial cells immobilized to a large extent on microcarriers.

From US-A 4,415,668 it is known that microcarriers, when brought into contact with organic solvents, shrink in their surface by up to 25%. This destroys the cell microcarrier interface and makes analysis by electron microscopy impossible. In order to avoid this, US Pat. No. 4,415,668 proposes suggest hydrophobizing the microcamer to avoid these disadvantages. Such microcarriers maintain their volume even in the presence of 70% ethanol. British patent application GB-A 2059991 discloses water-swellable microcarriers for cell cultivation. It is stated that the swelling capacity and the density of the particles in the water-swollen state are different for different microcarriers and can also be varied by crosslinking and thereby by the type of degree of crosslinking of the crosslinking.

The problems that arise during lyophilization or the cryopreservation of such microcarriers to which cells or biological material are bound are not discussed in the prior art. However, it has been shown that microcarriers coated with biological material are sensitive to freezing and thawing and that a considerable part of the biological material can be observed when thawing. The object of the invention is to eliminate this disadvantage and to provide an improved method and improved microcarrier which are largely insensitive to cryopreservation and show a reduced detachment of biological material immobilized thereon when thawed after freezing.

The object is achieved by a method for immobilizing biological material, in particular cells or cell assemblies on microcarriers, which is characterized in that the volume of the microcarrier to be coated is reduced by changing the osmotic conditions surrounding the said microcarrier (osmotic pretreatment) and then the microcarrier pretreated in this way is coated with the biological material in the usual way.

The volume reduction of the microcarriers results in a partial cross-linking of the carrier material, which makes it difficult for the microcarrier to swell again. The volume reduction is essentially largely irreversible. By adding crosslinking substances, such as glutardialdehyde or hexamethylene dioisocyanate or by other crosslinking processes, such as radiation, a further undesired increase in volume of the microcarrier pretreated by contraction can be reduced even further after water addition (chemical crosslinking of the microcarrier) after the osmotic pretreatment. .

It has been shown that in the course of cryopreservation, the detachment of biological material from microcarriers pretreated in this way is substantially less than if microcarriers not pretreated according to the invention are used. Since the microcarriers, which are preferably suitable for immobilizing cells and cell groups, usually have a water content of more than 60%, preferably more than 90%, it proves expedient to reduce the volume by removing water, preferably 50% or more of the water contained in the microcarriem to carry out. Such dehydration can be achieved by conventional methods for changing the osmotic conditions, such as, for example, the addition of dehydrating substances, the partial freezing of the solution containing the microcarriers or freeze drying (lyophilization).

The volume reduction can preferably be achieved by adding salt solution with at least 15% by weight of salt, preferably alkali metal halides or alkaline earth metal halides. An approximately 32% by weight sodium bromide solution (concentration in the total solution) is particularly preferably used for volume reduction, as a result of which maximum shrinkage of the microcarrier can be achieved. Also preferred additives are e.g. B. lithium bromide, sodium chloride or magnesium chloride. Organic additives which change the osmotic conditions according to the invention, dissolve in water in high concentrations but do not directly denature the microcarriers are also suitable. Such substances are, for example, dimethyl sulfoxide, acetone, methanol or ethanol.

In a preferred embodiment, in order to largely prevent or further reduce the swelling of the microcarrier, a substance is added after the osmotic pretreatment (preferably shortly after shrinking, preferably after about two minutes), which chemical crosslinking within the microcarrier (Crosslinking of the carrier material). Glutardialdehyde solution is preferably added to the microcarrier suspension. The concentration of the glutardialdehyde solution in the total suspension is preferably 0.01% by weight - 0.5% by weight. It is further preferred to incubate the microcarrier for a longer time in this solution before further use, temperatures between 0 and 10 ° C., preferably 4 ° C., being advantageous. A period of around 24 hours has also proven to be advantageous. It is further advantageous if, before coating the microcarrier with cells or cell groups, any free aldehyde groups present are saturated, for example by adding sodium borohydride. It is further preferred to prepare the pretreated microcarriers for the coating by washing with buffer solutions or cell culture media (e.g. keratinocyte medium and / or centrifugation) before the microcarriers are populated by cells or cell assemblies.

Such pretreated microcarriers usually have no more than 40% of their initial diameter. It has been shown that according to the invention all microcarriers can be used advantageously, the volume of which is preferably reduced by at least 50%. Such microcarriers only change their volume after coating with cells during the freezing process only insignificantly.

Another object of the invention is therefore the use of microcarriem with a water content of at most 50%, preferably at most 30%, particularly preferably at most 25% for immobilizing biological material, in particular cells. The water content of the microcarriers pretreated according to the invention fluctuates as a function of the initial water content. If, for example, the initial water content is above 90% and about 50% of the water is removed, the microcarriers pretreated according to the invention have a water content of about 40-50%. In the case of microcarriem, which have an untreated water content of approximately 60%, the water content can be significantly lower after the pretreatment according to the invention. Likewise, the reduction in water content can be varied by the duration of the incubation with the dehydrating agent and by the concentration of the dehydrating agent. It is preferred to carry out the volume reduction to such an extent that no significant volume reduction (for example at most by 10%) can be observed during the freezing process. A suitability according to the invention can therefore be readily determined by freezing the pretreated microcarriers, even without they being coated with cells. If the volume reduction during the freezing process is only slight (see above), it can also be assumed that the volume does not increase significantly (i.e. cell-detaching) when water is added. Due to the low water content, the microcarriers are volume-stabilized compared to dehydrating processes such as lyophilization and / or freezing / thawing cycles. Accordingly, the invention further relates to microcarriers which change (decrease) their volume by at most 10% after freezing and thawing.

In contrast to the method according to the invention, the addition of antifreezing agents alone when freezing (such as glycerol-ethylene glycol, hydroxyethyl starch) is not suitable to prevent the detachment of the cells. The microcarriers must be treated by the cells before coating.

Surprisingly, it has been shown that the microcarriers shrunk by osmotic pretreatment or lyophilization, when they are in aqueous solutions such as e.g. phosphate buffered saline (PBS), do not swell significantly again. It is advantageous that the microcarriers are treated for a long time to shrink. It has proven to be advantageous if the microcarrier treatment time is more than 10 hours, preferably more than 20 hours (eg 24 hours). An additional stabilization of the volume is achieved.

In a further preferred embodiment, to improve the coating of the microcarriers by cells (degree of coating), the microcarriers are coated after the osmotic pretreatment with substances which impart cell adhesion. Such substances are, for example, soluble collagen, fibronectin or similar substances.

The pretreated microcarriers are coated in accordance with the prior art, a method as in WO 96/12510 is preferably used, since this enables the microcarriers to be coated to a particularly high degree by cells or biological material.

Cells and cell assemblies such as Langerhans islands are suitable as biological material for coating the microcarriers. The coating is preferably carried out with epithelial cells, such as, for example, keratinocytes, fibroblasts, hepatocytes, islet cells or endothelial cells. Such cells are preferably proliferating cells and particularly preferably non-confluent cells which have the properties of basal cells. Such keratinocytes are described, for example, in WO 96/12510 and by S.T. Boyce, Surgery 103 (1988) 421-431, K.A. Hawboldt, J. Biotechnol. 34 (1994) 133-147, J.S. Tang, Clin. Orotope. 300 (1994) 254-258, N.A. Mufifty, Biotechnol. Proc. 11 (1995) 659-663.

Coating in the sense of the invention is not only to be understood as an immobilization of the cells or cell assemblies on the surface of the microcarrier, but also any type of immobilization of such cells and cell assemblies by microcarriers. If the microcarriers have a porous structure and the cells are correspondingly small, the cells can be, at least partially, embedded in the structure of the microcarriers. All microcarriers which contain liquid, preferably water, can be used as the microcarrier in the sense of the invention. Such microcarriers usually consist of crosslinked materials, preferably organic compounds, such as crosslinked polysaccharide, collagen, polymers of hyaluronic acid or hyaluronic acid esters (ethyl or benzyl ester) (Andreassi, L., et al., Wounds 3 (1991) 116-126 ; Myers, SR, et al., Abstract No. 85 in 5 ^th Annual Meeting of the European Tissue Repair Society (August 1995), Padua), HYAFF-11 ^® from FIDIA Advanced Biopolymeres, Abano T., Italy) fibrin-binding polypeptides, Dextran, gelatin, silicone, etc., or from a suitable mixture of different biocompatible materials. For example, Cytodex 3 ^® (cross-linked dextran matrix with a denatured collagen layer, 100-230 μm, Pharmacia AB, Sweden) can be used. Also suitable is Cellgen ^® (Koken Co., Japan), a reconstituted and cross-linked collagen made from bovine skin with a diameter of 200 - 500 μm or Cultispher-G ^® (macroporous microcarrier made from gelatin, Greiner, Germany), based on gelatin.

It is preferred that the size of the microcarrier after pretreatment is in the range between 10 μm and 2000 μm. Usually the microcarriers have a diameter of 50 - 500 μm. Microcarriers with a diameter of less than 10 μm are so small that they usually cannot be sufficiently coated with cells or other biological material. Microcarriers with a size of more than 2000 μm are so large that it is difficult to achieve a high coating rate. In addition, such microcarriers, particularly when used for transplantation, are not optimally suited. Large microcarriers cannot be optimally distributed evenly over a wound, and the detachment of the cells is also not uniform.

The cells which are used to coat the microcarrier can be allogeneic cells, autologous cells, mixtures thereof or groups of cells, preferably adherent cells such as keratinocytes, fibroblasts, endothelial cells, hepatocytes or islet cells.

In a preferred embodiment, the microcarriers are coated with cell adhesion-promoting substances, such as fibronectin or collagen, before coating, but preferably after the pretreatment according to the invention for shrinking. Coating with whole cells or fragments, such as membranes thereof, is also possible. Such a pre-coating is described for example in DE-A 26 51 685 and in WO 96/12510. The following examples, publications and illustrations further illustrate the invention, the scope of which is evident from the patent claims. The described methods are to be understood as examples which describe the subject matter of the invention even after modifications.

Unless otherwise noted, all percentages are percentages by weight.

FIG. 1 shows the typical course of the shrinkage of microcarriem with increasing salt concentration (sodium bromide)

FIG. 2 shows the shrinkage behavior of the microcarriers as a function of the crosslinking time (salt: sodium bromide).

Figure 3 shows the reduction in diameter, surface area and volume of the microcarriers with increasing salt concentration.

Figure 4 shows the temporal development of the degree of settlement of untreated, pretreated and collagen-coated beads. The degree of colonization was determined by counting the populated beads under the microscope at the appropriate time.

FIG. 5 shows the degree of colonization of beads before freezing and after

Thawing. The degree of colonization indicates the percentage to which a bead is colonized. The degree of settlement was determined by counting under the microscope. Is the degree of settlement e.g. 30%, this means that the bead surface is covered on average by 30% with cells.

Figure 6 shows the proportion of colonization before freezing and after thawing. The

Settlement percentage indicates the percentage of beads that generally have a settlement, regardless of the degree of settlement. A colonization proportion of, for example, 60% means that 60% of the beads present are colonized with at least one cell. example

Implementation of the shrinking and networking of microcarriers

5 ml of a suspension of Microcarriem (Koken Ltd., JP) (corresponding to approximately 1 million microcarriers) are placed in a sterile tube at room temperature. 20 ml of a 40% NaBr solution in PBS (phosphate buffered saline) are added (final concentration: 32% NaBr at room temperature). After approx. 1-2 min. 50.1 μl of 25% glutardialdehyde are added (final concentration: 0.05% glutardialdehyde). The homogeneous mixture is briefly shaken for homogeneous mixing and placed in the refrigerator overnight at about 16 h (4 ° C).

Then the carrier suspension at approx. 250 g for 10 min. centrifuged and the supernatant aspirated and replaced with 20 ml PBS. This step is repeated a total of three times, with the last step adding only 5 ml of PBS instead of 20 ml of PBS. To improve the settability of the microcarriers pretreated in this way, 500 μl of soluble collagen (Collagen S, Boehringer Mannheim GmbH, DE) are added to this suspension and this solution mixture is placed in the refrigerator overnight. The microcarriers are then centrifuged as described above and washed twice with the culture medium which is used to colonize the cells.

Based on their original diameter, the carriers have a diameter reduced by about 60% after this osmotic pretreatment. Optionally, the addition of glutardialdehyde can be dispensed with.

The change in the shrinking behavior of the microcarriers as a function of the salt concentration and thus of the osmotic conditions is shown in FIGS. 1 to 3.

FIGS. 4 to 6 show the degree of colonization and the proportion of colonization of the microcarriers as a function of time or as a function of freezing and thawing. Reference list

5 ^th Annual Meeting of the European Tissue Repair Society (August 1995), Padua

Andreassi, L., et al., Wounds 3 (1991) 116-126

Boyce, S.T., Surgery 103 (1988) 421-431

CA-A 1215922

DE-A 26 51 685

EP-A 0 242 305

EP-A 0 333 328

GB-A 2059991

Hawboldt, K.A., J. Biotechnol. 34 (1994) 133-147

Muffty, N.A., Biotechnol. Proc. 11 (1995) 659-663

Tang, J.S., Klin. Orotop. 300 (1994) 254-258

US-A 4,415,668

Williams, M.J., Cellular Physiology 136 (1988) 103-110

WO 89/03228

WO 93/23088

WO 96/12510

Claims

claims

1. A method for immobilizing biological material by coating on insoluble microcarriers, characterized in that the volume of the microcarrier to be coated is irreversibly reduced by changing the osmotic conditions surrounding the said microcarrier (osmotic pretreatment) and then the microcarrier thus pretreated is coated with biological material become.

2. The method according to claim 1, characterized in that cells, in particular epithelial cells or cell assemblies, are used as the biological material.

3. The method according to claims 1 or 2, characterized in that the volume of the microcarrier is reduced by the osmotic pretreatment to such an extent that there is no greater volume reduction during a freezing process.

4. The method according to claims 1-3, characterized in that the volume change takes place by dehydration.

5. Process according to claims 1-4, characterized in that the osmotic pretreatment results in a water withdrawal of more than 50% of the original water content.

6. The method according to claims 1-5, characterized in that the microcarrier consists of a cross-linked polysaccharide, collagen, dextran or gelatin.

7. The method according to claims 1-6, characterized in that the size of the microcarrier after the osmotic pretreatment is between 10 and 2000 microns.

8. The method according to claims 1-7, characterized in that the microcarrier is subjected to a chemical crosslinking after the osmotic pretreatment.

9. The method according to claims 1-8, characterized in that the microcarriers are coated after the osmotic pretreatment with substances which impart cell adhesion.