JP3134079B2 - Biological support for keratinocyte cultures constituted by thrombin-coagulated plasma proteins, as well as preparing said keratinocyte cultures, collecting and transferring said supports in transporting for therapeutic purposes Method used - Google Patents

Description

The present invention relates to a biological support for a cell culture consisting of a coagulated mixture of a plasma protein concentrate and thrombin, the preparation of a culture of keratinocytes. And the use of the support in the transport in the form of reconstituted epidermal tissue and the therapeutic application of the keratinocyte culture.

Prior art and its problems Laboratory reconstruction of simplified skin with the physiology of living or normal skin similar to human skin derived from several cells obtained from biopsy Is being studied intensively to replace skin that has been damaged by serious illnesses (such as hereditary) or destroyed by large burns.

Skin is a complex organ composed of three superimposed tissues. Epidermal tissue (85% of which is composed of keratinocytes forming an impermeable stratum corneum that isolates the body from the external environment); dermal tissue (separated by connective tissue consisting mainly of collagen) Subcellular tissue (including cells that become fat depots) beneath the dermal tissue. Thus, the artificial reconstruction of such a complex organ has a number of problems. The first tissue that was partially reconstituted in vitro was the dermal tissue, which was accomplished by Bell et al. (Proc. Natl. Acad. Scad.
ci., 76, 1979, 1274).

Starting from a skin biopsy, fibroblasts were successfully generated in culture initially in monolayer form. After repeated subcultures by suspending in a medium containing collagen (extracted from rat tail tendon), the monolayer forms a gel, allowing a three-dimensional culture. . In such cultures, the fibroblasts can be thought to interact with the collagen matrix, organize it, and shrink it, as in normal dermal tissue. This tissue reconstituted in vitro is known as "equivalent dermis tissue". After several weeks of growth, mechanical amounts of equivalent dermal tissue can be used to transplant into patients or injured humans. This transplant is not expected to cause rejection by the host. However, this equivalent dermal tissue is only a temporary treatment and cannot restore the barrier function of the skin.

In addition, H. Green et al. Have developed a method and medium that allow keratinocytes to grow over time (Proc. Natl. Acad. Sci., 76, 1979, 5665). This method inoculates keratinocytes dispersed with trypsin into a pre-made monolayer of lethally irradiated fibroblasts (especially 3T3 cells), which acts as both a vegetative layer and a matrix. By doing. Epidermal tissue grows rapidly to form tissue 3-5 cells thick. This layer can be implanted in the patient and differentiation can continue in situ. It has already been established that severe burn patients can be treated using this method (G.
G. Gallico et al., New England J. Med., 3
11,1984,448).

According to the method of Green et al., 1 m ² of epidermal tissue can be obtained from a 2 cm ² biopsy in 3 weeks.

Recovery of cells reconstituted to create a graft
Still has many technical problems. In fact, it is necessary to use enzymatic treatment to detach cells from the culture dish without the cells separating from each other. During this operation, regression of the cell layer is always observed, and thus some reduction of the graft surface area. Once the reconstituted tissue has been separated, it must be fixed to a support that allows the tissue to be transported and implanted into a patient. Vaseline-treated gauze dressings are commonly used. Such operations are cumbersome and time consuming.

Therefore, it would be very beneficial to use new biological supports that could be reabsorbed over time by the transplanted patient and that would simplify cell handling. Furthermore, in order to ensure its effectiveness, the support or its components must be able to be adapted for production and packaging by industrial processes.

Means for solving the problem Accordingly, Applicants have set forth a biological support for cell culture comprising a mixture of a concentrate of plasma proteins capable of coagulating with thrombin and the amount of calcium thrombin required to activate coagulation. Developed body.

Coagulation of plasma proteins in the presence of thrombin is primarily due to the formation of a polymerized fibrin network that mimics clot formation. Coagulation is carried out under conditions suitable for the formation of a film, in particular in a Petri flask or another flask suitable for cell culture, to form a support suitable for making cell cultures.

Concentrates of plasma proteins are indicated by the applicant in European Patent Application No. 884019613: Plasma proteins precipitate fresh plasma in two successive treatments with a 10% ethanol solution at 4 ° C. To be obtained. The concentrate contains 90% or more of fibrinogen and 1 g of protein.
Contains at least 0.1 IU of factor XIII and 0.03 to 0.1 g of fibronectin. The concentrate is packaged, lyophilized and stored until use.

The invention also relates to concentrates of proteins that can be coagulated by thrombin, especially protein concentrates packaged for the preparation of a biological support for cell culture.

At the time of use, the concentrate is redissolved in saline or in a solution containing a polyvalent protease inhibitor, preferably aprotinin, at a concentration of 3000 KIU / ml.

Thrombin is added in the presence or absence of calcium to activate the coagulation process and form a film that acts as a support for the cells. The process involves the conversion of fibrinogen to fibrin by the action of thrombin and XIII activated by calcium ions.
Includes the polymerization of fibrin monomer using fibronectin by the action of a factor.

In order to form a support according to the invention which is particularly suitable for cell culture, the thrombin concentration is preferably of the order of 10 IU / ml (this concentration is the case for the biological glue described in the aforementioned European Patent Application No. 884019613). , Which is much lower than the concentration used when it differs from the desired consistency).

According to another embodiment of the invention, various additives designed to promote cell proliferation, especially in vitro or in situ, and thus to promote healing of the wound after transplantation, can be incorporated into the support. .

Thus, the support may contain additional substances that promote cell growth, such as growth factors, especially EGF (epidermal growth factor).

The support of the present invention can also contain a repair agent, an antibiotic and the like.

The film-like support of the present invention is suitable for culturing cells that grow in a layer (or monolayer) on the surface. In particular, the film-shaped support of the present invention is particularly useful for preparing a human keratinocyte culture. These cells are derived from skin biopsies obtained from patients, and even in primary cultures that have undergone 1 to 4 passages at a dilution of 1/15 to 1/20, The cells may be stored in the form described above.

These keratinocytes made in the confluent layer are trypsinized and replaced in suspension in a suitable medium when the supports of the invention are inoculated with them.

The biological supports of the present invention are used in three different ways.

According to a first use, the biological support comprises
Produced in the form of a film by mixing the seed components in a culture dish; a suspension of keratinocytes is inoculated onto the film in a suitable medium. When the culture of keratinocytes becomes confluent or semi-confluent, the culture can be separated from the culture dish using forceps, and the patient can be used without the need for a temporary support such as gauze. Replacement tissue that can be directly recovered as a graft applicable to
ssue). This results in a considerable reduction in working time and 100% recovery of the grown tissue.

According to another method of using the support of the present invention, the two components of the support are mixed with a suspension of keratinocytes, and these cells are then incorporated into the formed film. According to this method, the two components can be mixed with the cell suspension in a culture dish and used as an implant in the same manner as described above; Blowing the biological support and keratinocytes with the vector gas (nitrogen) in the bar can be performed directly on the wound of a patient that has been treated to receive a graft.

According to another embodiment using the support of the present invention, the cells are formed by mixing the two components on a cell layer of keratinocytes pre-made in a culture dish. It is coated with a peelable and transportable film to be applied as an implant.

EXAMPLES The present invention will be described below with reference to Examples, but the present invention is not limited thereto.

Example 1-Preparation of a biological support for cell culture A biological support for cell culture comprises a concentrate of coagulable plasma proteins and the amount of calcium required to activate coagulation. Made by mixing with sex thrombin.

A. Preparation of Plasma Protein Concentrates The preparation of protein concentrates is described in European patent application no.
According to the method already described in 884019613. That is, using human plasma that does not cryoprecipitate, pH 7.2, temperature 4
Precipitation was repeated twice with a 10% ethanol solution at ° C. Between two successive precipitations, the purified product was subjected to a virus inactivation treatment. The precipitate is separated from the supernatant by centrifugation and
And then centrifuged again. The precipitate is replaced in a suspension of Tris / citrate buffer, the protein concentration is adjusted to about 35 g / L and the final concentration per gram of protein is
Lysine was added to 0.2 g. Diafiltration to remove alcohol and citric acid
After adjusting the ionic strength, the concentrate was filled in a flask and freeze-dried. This protein concentrate contains at least 0.9 g of fibrinogen, 0.03 to 0.06 g of fibronectin and 0.15 to 0.30 IU of factor XIII per gram of protein.

B. Preparation of Support for Cell Culture The above protein concentrate was prepared by adding 3000 KIU / ml (kallikrein inhibitor units / ml) in the presence or absence of aprotinin.
At the concentration of 1 in the aqueous suspension.

This solution is mixed with an equal volume of calcium thrombin (10 IU / m
l).

For a 10 cm diameter Petri dish, 2 ml of the protein suspension and 2 ml of thrombin were used, and the two solutions were simultaneously injected using two syringes interconnected by a mixing coupling. Shake the Petri dish to disperse it evenly and leave it for 15-20
Let stand for minutes. A film was formed over the Petri dish.

Culture dishes are “untreated for cell culture”
It is of the type and ensures that the support does not adhere permanently and therefore facilitates subsequent recovery.

The film was then covered with the cell culture medium. The above medium was changed several times until the osmotic pressure of the film stabilized within a range compatible with the physiological properties of the cells (ie 260-340 mosM).

Alternatively, the reconstituted protein concentrate can be dialyzed before mixing with thrombin.

Example 2-Making a keratinocyte culture on a biological support Obtained according to the classical method of Green et al. By skin biopsy obtained from the skin of a patient (or fetal skin forming a fetal cell bank). Primary cultures of the obtained keratinocytes were used. These primary cultures can be passaged 4-5 times at 1/10 dilution.

The layer of confluent keratinocytes was trypsinized, replaced with a suspension in medium, and inoculated at 1/10 dilution into a Petri dish covered with a film of the biological support obtained in Example 1.

After several hours, the cells attached to the support and grew normally there, forming fragments of confluent epidermal tissue with a thickness of 3 or 4 cell layers.

The above fragments of reconstructed epidermal tissue are attached to a support and can be peeled from the culture dish using forceps and applied directly to a wound that has been prepared to receive a graft.

Because the cells attach to the support, there is no need to attach the reconstituted epidermal tissue to another support, such as petrolatum-treated gauze, which must be used in other types of cultures. This considerably reduces the working time. That is, while the conventional method can process only 4 boxes, it can process 40 boxes.

In addition, the support is well tolerated in working processes, does not shrink when peeled off, and can recover 100% of the cell layer surface area of the culture.

Example 3-Recovery of reconstituted cell layer using biological support Inoculation of keratinocytes in a Petri dish coated with a layer of lethally irradiated fibroblasts according to the conventional method of Greene did.

When the keratinocyte sheet became confluent and formed several cell layers, the medium was removed, an EDTA solution was added over 1.5 hours, and washed twice with PBS. The biological support was then poured directly onto the cell layer according to the method of Example 2.

If a film is formed covering the cells, the film can be peeled off from the culture dish using tweezers and used as an implant, as in the above-described embodiment.

Example 4 Incorporation of Cells into Biological Support A syringe of a protein solution and a syringe of a thrombin suspension containing keratinocytes were provided. Keratinocytes can be obtained from fresh trypsinized cultures or from cell banks stored in liquid nitrogen.

The two syringes described above were interconnected by a mixing coupling, and the support containing keratinocytes was sprayed onto a Petri dish (or the wound to be implanted); the cells were preserved in the film during coagulation. Spraying can be performed with vector gas (2-2.5 bar nitrogen gas).

This spray did not denature the cells and the cell layer was observed to be repaired in the in vitro culture. Thus, when the above mixture is sprayed directly onto the wound in a very thin layer, the cells grow normally or almost normally.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-2-114 (JP, A) US Patent 4,642,120 (US, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 5 / 00-5/28 A61L 27/00 C12M 3/00-3/10 BIOSIS (DIALOG) WPI (DIALOG) (54) Title of the Invention Biological support, as well as methods of using the above support in preparing, recovering and transferring keratinocyte cultures for therapeutic purposes

Claims (15)

(57) [Claims] (1) per 1 g of protein obtained by treating plasma with ethanol: at least 0.9 g of coagulable fibrinogen, 0.1 IU of factor XIII, and 0.03-
(2) about 10 IU / m2 of thrombin coagulating plasma protein concentrate containing 0.1 g of plasma fibronectin and (2) the solubilized protein concentrate
A biological support for the culture of keratinocytes, characterized by being formed by mixing with a proportion of calcium thrombin in l. 2. The biological product according to claim 1, wherein the protein concentrate is obtained by precipitating fresh plasma in two successive treatments with a 10% ethanol solution at 4 ° C. Support. 3. The biological support according to claim 1, wherein the protein concentrate is freeze-dried. 4. The biological support according to claim 1, wherein the protein concentrate is suspended in an aprotinin solution. 5. The biological support according to claim 1, further comprising a cell growth enhancer as an additional substance. 6. The biological support according to claim 1, further comprising an antibiotic as an additive. 7. The biological support according to claim 1, further comprising a substance that promotes wound healing as an additional substance. 8. Replacement tissu which is directly recoverable, transportable and applicable as an implant.
A method of using a biological support according to any of claims 1 to 7 in producing a culture of fetal or adult human keratinocytes capable of forming e). 9. A method of mixing two components of a biological support so as to form a uniform film in a culture dish, and inoculating the film with a suspension of keratinocytes in a medium. 9. The method according to claim 8, wherein: 10. The method according to claim 8, wherein the two components of the biological support are mixed with a suspension of keratinocytes and the cells are then incorporated into a film to be formed. . 11. The method according to claim 9, wherein a keratinocyte suspension is obtained after dispersion of a fresh cell layer prepared in advance. 12. The method according to claim 9, wherein the keratinocytes are obtained from a cell bank stored in liquid nitrogen. 13. The method according to claim 1, wherein the collection and transfer of a pre-made culture of human (fetal or adult) keratinocytes.
A method using the biological support according to any one of claims 1 to 7. 14. The method according to claim 13, wherein the two components of the biological support are mixed on a previously prepared cell layer in a culture dish. 15. A concentrate of a protein coagulable by thrombin, which is packaged for the preparation of a biological support according to claim 1.