HK1070099A - Keratinocytes which may be used as biologically active substances for the treatment of wounds - Google Patents

Description

Keratinocytes as biologically active substances in the treatment of wounds

Field of the invention

The invention relates to a novel keratinocyte which can be cultured in vitro and an application thereof in preparing medicines for treating acute and chronic wounds. The present invention therefore belongs to the field of medicine, in particular to the field of wound healing by tissue engineering.

Prior Art

Allogeneic (allogenic) keratinocytes have been used successfully for a long time in the treatment of wounds, in particular in the treatment of ulcers and/or burn wounds (Maier, 1993; Sch * nfeld et al, 1993). Wounds that are resistant to the traditional treatment methods of long cycle length are often treated effectively with allogeneic keratinocytes (Phillips and Gilchests, 1993; Beele et al, 199 l; Leigh et al, 1991; Lindgren et al, 1998). When using allogeneic keratinocytes, attention was initially focused mainly on the renewal of the skin, i.e. on the regeneration of the transplanted epidermis. However, recent studies have shown that the success of treatment is mainly based on stimulation of the body's own re-epithelialization, and not on the "growth" of the allogenic grafts in the wound. Recently, many investigations have shown that transplanted keratinocytes remain in the wound for a specific period of time and are then eliminated from the body in a clinically imperceptible manner (Kawai et al, 1993). The stimulation of the body's own wound healing mainly results from the optimized release of allogeneic keratinocytes in space and time: a diverse group of growth factors, cytokines, extracellular matrix (ECM), small molecules and proteases (Lang et al, 1996; Marcusson et al, 1992). The complexity and amount of factor released confirms that this treatment is superior to traditional monotherapies, such as those employing isolated growth factors. However, in addition to the main effects of re-epithelialization, treatment with keratinocytes can produce macroscopic and microscopic changes in granulation tissue (Lang et al, 1996). Another frequently described benefit of keratinocytes in patients is a marked analgesic effect after transplantation (Sch * nfeld et al, 1993).

For wound healing, it is advantageous to use undifferentiated, actively dividing keratinocytes, because actively dividing keratinocytes have a complex secretion profile (profile) which promotes wound healing. In addition to stem cells, undifferentiated keratinocytes lose their proliferative potential in vivo only after a few cell divisions during natural differentiation; this phenomenon has been observed to date in vitro during the culture of keratinocytes (Barrandon and Green, 1987). As a result, actively dividing keratinocytes which are particularly suitable for wound healing have hitherto only been cultured in vitro, which has made their use in medical terms difficult and expensive.

The allogeneic keratinocytes used in the treatment may be either directly in the form of so-called "keratinocyte sheets", which consist of a multilayer, enzymatically detachable cell aggregate, or may be combined with a biocompatible carrier to form a "bioactive wound healing dressing". The latter has the advantage that it does not need to be detached from the culture dish substrate by enzymatic pre-treatment before use. Furthermore, the use of biocompatible membranes as carriers for the cultivation of keratinocytes (EP 0462462, US 5,658,331; US 5,693,332; EP 0518920) makes it possible to prepare biologically active wound-healing adjuvants more rapidly (earlier), since the cells no longer have to form self-contained cell aggregates. Vectors that have been grown to a sub-confluent state are useful for wound treatment. In addition to being rapidly available, an additional advantage of using sub-confluent cell aggregates is the less vigorous differentiation of the correspondingly cultured keratinocytes, which is advantageous for wound therapy.

Keratinocytes can be cryopreserved at-30 ℃ to-196 ℃ (preferably-70 ℃ to-90 ℃) directly or together with biocompatible carrier materials and then used for wound healing without any harmful effects (De Luca et al, 1992; Teepe et al, 1993). This further increases the therapeutic usefulness of keratinocytes, which makes it possible to maintain to some extent the supply of biologically active wound healing adjuvants.

Problems to be solved by the invention

The keratinocytes disclosed in the prior art and the biologically active wound-healing adjuvants produced therefrom have specific drawbacks, which the present invention aims to overcome.

The obvious disadvantages of the primary keratinocytes of origin known today are: the cells can only self-replicate for a few generations in an in vitro cell culture process without losing their high division potential and hence their suitability for the preparation of biologically active wound healing adjuvants. According to the prior art, the person skilled in the art can only increase the cell number by about 10 by in vitro culture³To 10⁴And (4) doubling.

Another drawback that follows is that the limited in vitro culture capacity of the keratinocytes leads to the necessity of frequent and complex re-separations; this means that the resulting replicated cellular material is not uniform and thus the wound healing dressing produced therefrom will, or at least likely will, be of varying quality.

A further disadvantage is that re-isolation of keratinocytes from various donors will result in an increased risk of infection for the recipient individual of the wound healing colony. For example, the risk of infection with HIV or hepatitis is increased.

Description of the invention

The invention relates to keratinocytes having a high proliferation potential, which cannot be propagated indefinitely but can be replicated at least 150 times by means of in vitro culture. Which results in a cell proliferation factor of about 10⁴⁴. The corresponding keratinocytes still retain their advantageous properties for the treatment of wounds.

The keratinocytes of the invention are mainly keratinocytes isolated from a donor and cultured in vitro, which can be carried out by any person skilled in the art according to the methods disclosed, for example, by Rheinwald and Green in 1975.

The present invention preferably relates to a keratinocyte cell isolated from the epidermal portion of a foreskin. Human-derived keratinocytes, in particular keratinocytes in culture KC-BI-1, which are deposited under the Budapest treaty for patent purposes at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of microorganisms and cell cultures), GmbH, Braunschweig, Germany, on 27 th 6.2001, with the deposit number DSM ACC2514, are preferred. The present invention also includes those keratinocytes derived from KC-BI-1 culture (DSM ACC 2514). Thus, the present invention also includes all cells and cultures obtained from the re-passaging and/or re-cloning of the original KC-BI-1 culture.

The culture of keratinocytes of the present invention is exemplified by the culture of keratinocytes KC-BI-1 (example 1). The use of complex media, as detailed in example 1, and of feeder cells, in particular murine 3T3 fibroblasts treated with lethal radiation, will facilitate the culture. The amount of fetal bovine serum should be 2% to 10%. The trophoblasts can be prepared by methods well known to those skilled in the art, such as the method described in example 2. The re-culture of keratinocytes of the invention is particularly advantageous when the maximum confluence is 80%. The keratinocytes are cultured at a temperature of between 35 ℃ and 38 ℃, preferably at a temperature of 37 ℃, with a relative humidity of > 90%, preferably 95%, and CO₂The saturation is 5% to 9%.

According to the culture process described in example 1, keratinocytes, in particular human keratinocytes derived from the epidermal part of the foreskin, have a population doubling time of 1 to 2 days (fig. 1). These cells were able to replicate at a substantially constant replication rate for many generations (FIG. 2). However, the present invention is not limited to only the keratinocyte KC-BI-1, and those skilled in the art can implement the present invention using any keratinocyte described in claims 1 to 8 under appropriate conditions.

The term "incapable of immortalization" in the sense of the present invention means that the keratinocytes obtained by primary isolation and cultured therein cannot be transformed spontaneously and/or are not transformed by molecular biological, chemical or physical methods known in the present research. The latter means that the cells have not been treated with, for example, viral factors or sequences, chemical mutagens, or, for example, with radiation or a combination of different methods.

A keratinocyte cell that is "incapable of immortalization" is also defined as having no or substantially reduced telomerase activity as compared to a transformed tumor cell line (H.die. rle-Bachor and Boukamp, 1993) or an immortalized cell line, particularly a HeLa cell line (see FIG. 3). This is also true in particular in comparison with the immortalized keratinocyte cell line HaCat.

The term "incapable of immortalization" also means that said keratinocyte cell line is incapable of replicating like, for example, immortalized keratinocytes in the absence of fetal bovine serum and/or in the absence of trophoblasts and/or in the absence of the epidermal growth factor EGF (Schoop et al, 1999).

However, the term "incapable of unlimited proliferation" also means that the keratinocytes do not change their characteristic phenotype when the replication of the cells is increased (see FIG. 4).

"unable to proliferate indefinitely" also means that the keratinocytes show a normal differentiation spectrum after transplantation into nude mice, in particular BALB/c. "Normal differentiation potential" as used herein refers to the ability of keratinocytes to develop into terminally differentiated keratinocytes and form the basal epithelial layer (epithelial epidermal layer) and stratum corneum (stratum corneum) in the same manner as autologous keratinocytes.

The invention also relates to keratinocytes which are not immortalized but are capable of replicating at least 200 passages by in vitro cell culture. The invention also relates to keratinocytes that are not immortalized but are capable of replicating at least 250 passages by in vitro cell culture. The invention also relates to keratinocytes which are not immortalized but are capable of replicating at least 300 passages by in vitro cell culture.

The advantage of the present invention is that the keratinocytes are replicated from one or only a few donors. For example, 10 can be obtained after 150 rounds of cell replication starting from one donor⁴⁴10 cells obtained after 250 rounds of cell replication⁷⁷10 cells can be obtained after 300 rounds of cell replication⁹⁰And (4) cells. Thus, the present invention for the first time makes it possible to produce large quantities of standardized cellular material for the preparation of stable and quality reliable bioactive wound healing aggregates. A corresponding amount of standardized cellular material can be replicated, for example, starting from a refrigerated cell bank prepared from keratinocytes having the properties described in the present invention.

By using standardized cellular material as starting material for the preparation of a bioactive wound healing dressing, the risk of infection of a possible recipient individual is also reduced, since the isolation of keratinocytes will be limited to a few donors, preferably one donor.

The present invention therefore overcomes the serious drawback of the hitherto known keratinocytes being passable only to a limited extent.

The invention also relates to a product consisting of a support covered with keratinocytes according to the invention. By "covering" is meant for the purposes of the present invention that the surface of the carrier is partially or completely populated with keratinocytes of the present invention. Partially colonised vectors will be particularly suitable because of the reduced incubation time required before the vector can be used in wound therapy.

The carrier suitable for the purposes of the present invention is characterized in that it is a biocompatible carrier material which can be used for the preparation of pharmaceutical compositions. For example, hydrophobic biocompatible carrier materials as described in WO 9113638 may be used. However, those support materials having a significantly hydrophilic character may also be employed.

A preferred embodiment of the present invention comprises the use of a carrier material consisting of an esterified hyaluronic acid polymer. In a particularly preferred embodiment, an esterified hyaluronic acid polymer is used, which consists of a porous polymer membrane with a defined geometry. For example, the polymer film has a thickness of 10 to 500 μm, is opened with pores having a pore diameter of 10 to 1000 μm, has a definite, uniform size, and is formed in an ordered arrangement with a constant interval of 50 to 1000 μm from each other. Such a film is described in EP 0462426. Porous carrier materials are particularly suitable because they do not require the bioactive wound healing dressing to be placed in a particular orientation on the wound. An esterified hyaluronic acid porous carrier matrix with a specific geometry and colonized by keratinocytes of the invention is described by way of example in example 3. The carrier matrix is produced by MessrsFidia Advanced Biopolymers Ltd, Abano Terme, Italy, under the product name "Laserskin" in the Germany market.

Suitable properties of this carrier material which are particularly preferred in the production of biologically active wound-healing dressings in conjunction with keratinocytes have been demonstrated in animal models (Lam et al, 1999) and in humans (Harris et al, 1999). In addition to improving migration and differentiation of epithelial cells, matrices composed of hyaluronic acid esters have a positive effect on both angiogenesis and collagen production. However, currently used wound dressings with hyaluronic acid esters as the matrix, all covered on their surface with autologous keratinocytes and/or skin equivalents of complex structure obtained from keratinocytes and fibroblasts. Therefore, it has the above-mentioned drawbacks of the prior art. This drawback will be overcome in particular with the beneficial allogeneic keratinocytes of the invention.

Another preferred embodiment of the invention relates to a product comprising keratinocytes of the invention and a resorbable polymer, consisting of: polyesters, polycarbonates, polyanhydrides, polyorthoesters, polyglycopeptides (polydepsipeptide), polyetheresters, polyamino acids or polyphosphazenes, in particular poly (L-lactide), poly (D, L-lactide), poly (L-lactide-co-D, L-lactide), poly (glycolide), poly (L-lactide-co-trimethylene-carbonate) or poly (dioxane). These polymeric substances can be either porous or non-porous materials.

The invention also relates to a method for the cryopreservation of the keratinocytes of the invention at temperatures between-20 ℃ and-196 ℃, preferably below-180 ℃. The keratinocytes may be frozen using standard methods well known to those skilled in the art. DMSO is particularly useful as a cryoprotectant. Other cryoprotectants, such as glycerol, hydroxyethyl starch, or a combination of the two, and combinations of these with DMSO, may also be employed. Suitable processes are described, for example, in WO9624018, US 5891617 or US 5298417.

The invention also relates to the cryopreservation of a support covered with keratinocytes according to the invention, characterized in that the keratinocytes and their corresponding support are cryopreserved at a temperature of-20 ℃ to-196 ℃, preferably-60 ℃ to-80 ℃. The advantage of cryopreservation is that the resulting bulk product can be stored so that its homogeneity of quality can be checked by random sampling before clinical use. Finally, storage ensures that wound healing aggregates are obtained in a short time for medical purposes.

Suitable cryoprotectants for the products of the invention are hydroxyethyl starch, for example in an amount of 7 to 13% (w/w). However, it is also possible to use DMSO or glycerol in combination with various cryoprotectants, in particular hydroxyethyl starch, DMSO and/or glycerol. Trehalose may also be used as a cryoprotectant.

After a rapid decrease of the temperature from 37 ℃ to-5 to-10 ℃ in 2-5 minutes, preferably to-6 to-8 ℃, the product containing the carrier and the keratinocytes of the invention will be equilibrated at the appropriate temperature for 15-30 minutes, preferably for 23-26 minutes. The product is then frozen, for example to-60 to-80 ℃ at a freezing rate of < 1 ℃/min, preferably 0.2 to 0.6 ℃/min, most preferably 0.4 ℃/min.

Example 4 describes the cryopreservation of hyaluronic acid ester carrier matrix coated with KC-BI-1 by specific examples. However, other cryopreservation methods may be employed, such as those described in WO 95707611, WO9624018, EP 0296475; the above list cannot be considered as an exhaustive list of preservation methods, but merely indicates that the method of cryopreservation of a product consisting of a biocompatible carrier and keratinocytes is part of the state of the art.

The invention also relates to the medical use of the keratinocytes described in the invention and/or the products of said keratinocytes and the vectors described herein, in particular in the treatment of wounds. A particular embodiment of the invention is the use of the keratinocytes of the invention and/or the products of said keratinocytes and a carrier for the treatment of burns and/or ulcers. The burn to be treated is preferably a second degree burn, and the ulcer is preferably a difficult-to-heal chronic lower limb ulcer, an Ulcus cruris type ulcer, an Ulcus cruris venosum type ulcer or a diabetic ulcer, and a decubitus ulcer.

Said medical use comprises the use of said keratinocytes and/or the product consisting of the keratinocytes and the carrier according to the invention in combination with and/or as a supplement to the traditional treatment methods known in the art for the effective healing of wounds. This means the combined and/or complementary use of one or more other substances effective in healing wounds. It may be mentioned that in the treatment of Ulcus cruris venosum hydrocolloids may be used additionally and/or in combination and/or in addition antimicrobial substances, e.g. antibiotics, may be used.

The invention also relates to the use of the keratinocytes and/or the biocompatible carrier according to the invention and the products formed by said keratinocytes for the preparation of a medical product for the treatment of wounds, in particular for the treatment of burns and/or ulcers, for example for the treatment of second degree burns, Ulcus cruris (venosum), diabetic ulcers, or decubitus ulcers.

The invention also relates to a method for treating the wound described above, which method is characterized in that the keratinocytes according to the invention and/or the product according to the invention comprising keratinocytes and a carrier are applied to the wound in need of treatment. The keratinocytes and the product may be either fresh or after cryopreservation. A corresponding method of treating a wound is described in example 5.

Drawings

FIG. 1: replication of keratinocytes KC-BI-1, expressed as a function of culture time.

This figure shows the number of cell replications (CPD ═ cumulative population doublings) of keratinocytes KC-BI-1 in the 94 day culture phase. The cells were approximately doubled 75 times over the 94 day observation period. This corresponds to an average doubling time of 1.25 days per round of cell replication, or 12.5 passages doubling every 10 days.

FIG. 2: doubling of keratinocytes KC-BI-1 in 10 months.

The figure shows the cell replication of keratinocyte KC-BI-1 over 10 months, expressed as a function of Population Doubling (PD) versus 1-67 cell generations.

FIG. 3: relative telomerase activity was determined.

This figure shows a comparison of telomerase activity in keratinocytes KC-BI-1 after passage 1, 12, 18, 40, and 57 with that in the HeLa cell line. This figure shows that keratinocytes KC-BI-1 have little or only slight telomerase activity compared to the immortalized HeLa cell line.

FIG. 4: morphology of keratinocyte KC-BI-1 after passage 5 and 60.

The keratinocytes KC-BI-1 at passage 5 (culture time 25 days) and at passage 60 (culture time 300 days) were not morphologically different from each other when observed under an optical microscope.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1: the method for culturing the keratinocytes of the invention is described in detail by taking the culture KC-BI-1(DSM ACC2514) as an example

1. Material

Keratinocyte KC-BI-1; irradiated 3T 3-murine fibroblasts (trophoblasts, for their preparation see example 2); cell culture medium K/1 (composition as follows); EDTA (0.02%); Trypsin/EDTA (0.05%/0.01%)) (ii) a Cell culture flasks (T-cell flasks): 25cm²，80cm²，175cm²。

2. Cell thawing

2.1 thawing of trophoblasts

Cells were quickly thawed and then added to 5-10ml of pre-warmed K/1 medium. The corresponding number of feeder cells was transferred to a suitable cell culture flask, to which K/1 medium was added:

25cm²t-cell flask 0.5X 10⁶Cell culture medium final volume: 5-6ml

80cm²T-cell flask 1.5X 10⁶Cell culture medium final volume: 20ml of

175cm²T-cell flask 3.5X 10⁶Cell culture medium final volume: 50ml of

The feeder cells can be immediately taken into use or within 24 hours.

2.2 keratinocyte thawing

Cells were quickly thawed and then added to 5-10ml of pre-warmed K/1 medium. The corresponding number of cells was added to the cell culture flask containing the feeder cells, on which fresh medium was added:

25cm²t-cell flask 0.15X 10⁶A cell; final volume of culture medium: 6-10mL

80cm²T-cell flask 0.4X 10⁶A cell; final volume of culture medium: 20mL of

175cm²T-cell flask 1X 10⁶A cell; final volume of culture medium: 50mL

3.0 cultivation

The cells are incubated at 35-39 deg.C, preferably 37 deg.C. Relative humidity of> 90%, preferably 95%, CO₂The concentration is 5-9%. At 80% of maximum confluence, the keratinocytes were re-cultured.

For this purpose, the cell culture supernatant was discarded. The feeder cells were rinsed twice with 0.02% EDTA (2-10ml) and incubated at 37 ℃ for 5-10 minutes before being detached from the cell culture flask by tapping (or shaking). The keratinocytes were then treated with trypsin/EDTA (0.05%/0.01%, 1-6ml) for 5-10 minutes at 37 ℃ and carefully detached by tapping. The remaining cells were carefully scraped off with a cell scraper if necessary. K/1 medium was added to neutralize the trypsin/EDTA solution and the cells were dispersed by carefully blowing up and down. Cells were seeded in the numbers indicated in section 2.2. Cell culture medium K/1 was changed on day 3, then every 2 days.

4.0K/1 preparation of the Medium

All ingredients and stock solutions were added and mixed in the order shown in table 1. WFI (Water for injection) was added to make up the mixture to 1 liter. Then adjusting the pH value to 7.0-7.2 by NaOH or HCl. The osmolality should be 320-400 mOsm/kg. Finally, the K/1 medium is sterilized by filtration.

4.1 preparation of stock solutions

Triiodothyronine

13.6mg of triiodothyronine was dissolved in 1ml of 0.1NaOH solution, and 99ml of PBS was added thereto. The final solution was diluted 1: 100 with PBS. 1ml of this solution per liter of medium must be added.

EGF

1mg of EGF was dissolved in 100ml of WFI. 1ml of this solution per liter of medium must be added.

rh-insulin (soluble only at pH < 4.0)

To 0.9L of WFI was added 5g of rh-insulin and the pH was adjusted to 2.5 with 6M HCl. After the rh-insulin was dissolved, the pH was adjusted to 8.0 with 1M NaOH. The mixture was made up to 1 liter with WFI. 1ml of this solution per liter of medium must be added.

4.2 composition of the culture Medium

Composition (I)	concentration/L
		WFI (Water for injection)	0.8L
Glutamine-containing DMEM	10.035g
		HAM’s F12	2.66g
Sodium bicarbonate	3.07g
		Pyruvic acid Na	0.11g
Apo-transferrin	5mg
		Semi-sulfated adenine	147.4mg
Maohuosu (soluble in several drops of DMSO)	2.05mg
		Rh-insulin concentrate	1.0mL
Hydrocortisone 10mM	0.11mL
		Triiodothyronine stock solution	1.0mL
EGF-stock solutions	1.0mL
		Phenol Red	8.1mg
FCS(2-10％)	20-100mL

6M HCl	Adding the required amount of the raw materials
		40％NaOH	Adding the required amount of the raw materials
WFI	Adding to 1.0L

However, the cells may also be cultured from a fresh biopsy, for example, from the epidermal part of the foreskin. Initial isolation of undifferentiated, proliferating keratinocytes was performed using the method described by Rheinwald and Green in 1975.

The feeder cells used may be, for example, the cells described in example 2. Other lethal fibroblasts, preferably other murine fibroblasts, and most preferably progeny of the cell line 3T3, may also be used.

Example 2: preparation of irradiated 3T3 feeder cells for keratinocyte culture

1. Material 1

Murine 3T3 fibroblasts (e.g., ATCC CCL 92, 3T 3-swiss albino, contact-inhibiting fibroblasts) can be obtained from the American Type Culture Collection (ATCC), 10801 university boulevard, Manassas, VA, USA; DMEM + 10% Fetal Calf Serum (FCS); PBS; 0.2% trypsin solution; 0.04% EDTA solution; cell culture flasks (T-cell flasks): 25cm²，80cm²，175cm²。

2. Cell thawing

Cells were quickly thawed and then added to 5-10ml of pre-warmed K/1 medium. The culture medium containing DMSO was removed by centrifugation. The cells are suspended in 5-10ml of medium. After cell counting, the cells were seeded in a suitable cell culture flask at a seeding density of 10³To 10⁴Cells/cm². Then, the temperature is kept at 35-39 ℃, preferably 37 ℃. Relative humidity > 90%, preferably 95%, CO₂The concentration is 5-9%.

3. Cell culture

The growth of the cells was observed daily. The cell density cannot exceed 70-80% of the maximum confluence. Subculture was performed every 2 to 4 days as needed. For this purpose, the medium was discarded and the cells were rinsed with a suitable volume (0.5-5ml) of a 1: 2 mixture of EDTA and trypsin. The detached cells are then added to 3.5-20ml of culture medium. At 10³To 10⁴Cells/cm²Is carried out at the seeding density.

4. Irradiating the trophoblast

Trophoblasts are administered at about 60Gy (¹³⁷6000 rad in Cs radioactive sources) receives radiation.

Both irradiated and non-irradiated cells were frozen in liquid nitrogen using standard methods and stored for extended periods of time.

Example 3: method for coating a carrier matrix (Laserskin in this case) with keratinocytes from culture KC-BI-1(DSM ACC2514)

The preparation of the bioactive wound healing dressing of the present invention will now be exemplified. The wound healing dressing described herein consists of keratinocytes of the invention, KC-BI-1, and Laserskin, which is a bioresorbable carrier matrix containing hyaluronic acid esters.

However, the scope of the invention is not limited to the combinations described herein. Any keratinocyte cell having the novel properties of claims 1-8 can be used for covering.

Other suitable carrier matrices may also be employed, provided that they are biocompatible carrier materials that can be used in the preparation of pharmaceutical compositions. For example, hydrophobic biocompatible carrier materials as described in WO 9113638 may be used. In addition, support materials having a pronounced hydrophilic character may also be employed.

Another preferred embodiment of the present invention comprises the use of keratinocytes of the invention in combination with a resorbable polymer comprising: polyesters, polycarbonates, polyanhydrides, polyorthoesters, polyglycopeptides (polydepsipeptides), polyetheresters, polyamino acids or polyphosphazenes, in particular poly (L-lactide), poly (D, L-lactide), poly (L-lactide-co-D, L-lactide), poly (glycolide), poly (L-lactide-co-trimethylene-carbonate) or poly (dioxane), and also perforated films made of the above-mentioned polymers.

1. Material

K/1 medium (see example 1); PBS; 0.04% EDTA (diluted to 0.02% with PBS); trypsin/EDTA (0.05%/0.01%); sterile Roux dishes (T25 cm)²，T 80cm²，T175cm²B) of the group A and B); placing in 144X 21Petri dish (surface area 145 cm)²) Laserskin (messrs. fidia Advanced Biopolymers srl, abaco term, italy); 3T3 nourishingA cell; the keratinocytes of the present invention are, for example, KC-BI-1.

2. Culture of bioactive wound healing dressing

2.1. Material

Irradiated feeder cells, e.g., murine 3T3 fibroblasts mentioned in example 2; stored keratinocytes of the invention; laserskin (final product) with size of 8.5cm × 8.5cm placed in Petri dish; k/2 culture medium.

2.2.3 inoculation of feeder cells (seeding out) with T3

Trophoblasts prepared according to example 2 were placed on Laserskin at a seeding density of about 15,000 to 25,000 cells/cm²(approximately corresponds to 3X 10⁶cell/Petri dish). The Petri dishes were then incubated at 35 to 37 ℃ with a relative humidity of > 90% and CO₂Concentration 5-11% (preferably CO)₂Concentration 7-9%) in an incubator at 37 ℃. Keratinocytes were seeded on the trophoblast colonies on the same day, or at the latest on the following day (after 24 hours).

2.3. Inoculation and culture of keratinocytes

The keratinocyte is adopted to prepare the bioactive wound healing dressing. For example, subculture of keratinocytes can be carried out as follows:

0.02% EDTA (80 cm)²Roux petri dish: 8 mL; 175cm²Roux petri dish: 10ml) the sub-confluent cultures were rinsed. Then, the feeder cells were treated with 0.02% EDTA (80 cm)²Roux petri dish: 8 mL; 175cm²Roux petri dish: 10ml) was incubated at 37 ℃ for 5-10 minutes and carefully shaken off.

Keratinocytes were dissolved in a mixture of trypsin/EDTA (0.05%/0.01%) as described in example 1 (80 cm)²Roux petri dish: 2-3 mL; 175cm²Roux petri dish: 5-6ml), followed by addition of cell culture fluid(80cm²Roux petri dish: 7-8 mL; 175cm²Roux petri dish: 14-15ml) and cells were dispersed by careful pipetting up and down.

The keratinocytes of the invention are placed on a Laserskin membrane with feeder cells and seeded at a density of about 15,000 to 25,000 cells/cm²(approximately corresponds to 3X 10⁶cell/Petri dish). The cells are then cultured at 35-39 deg.C, preferably 37 deg.C, to 30-100% confluence, preferably 80-100% confluence. Relative humidity > 90%, preferably 95%, CO thereof₂The concentration is 5-9%.

Example 4: the invention relates to a freezing preservation method of a bioactive wound healing dressing

After the keratinocytes have settled on the carrier substrate until 30-100% confluence, preferably 80-100% confluence, the product of the invention can be placed in a suitable container (e.g. a heat-sealed PP bag) and frozen under controlled conditions. For this purpose, the medium was carefully removed and replaced with 20ml of K/2 frozen medium at 2-6 ℃. Subsequently, the product was packaged under aseptic conditions and frozen according to the following method:

the temperature is rapidly lowered to-5 to-10 c, preferably-6 to-8 c, within 2-5 minutes, and the product is then equilibrated at the respective temperature for 15-30 minutes, preferably 23-25 minutes. The product is then cooled, for example to-60 to-80 ℃ at a freezing rate of < 1 ℃/min, preferably 0.2 to 0.6 ℃/min, most preferably 0.4 ℃/min. The product is stored at-60 to-80 ℃.

K/2 freezing medium:

k/1 growth medium (see example 1) was mixed with 7-13% (w/w) hydroxyethyl starch.

Example 5: the use of a support matrix colonising keratinocytes according to the invention for covering wounds, in the case of venous leg ulcers, is illustrated by way of example

1. Transport of freshly prepared wound healing dressings

When keratinocytes are growing on LaserskinAfter reaching 30-100%, preferably 80-100% confluence, the culture is rinsed 1 or more times with the appropriate amount (preferably 30ml) of K/3 transport medium. The bioactive wound healing dressing is transferred to an appropriate amount (preferably 20ml) of K/3 transport medium. The headspace of the Petri dish was rapidly filled with CO mixed with 5-10%₂Then sealed with an adhesive tape, such as Parafilm, and then placed in a shipping box for immediate delivery to clinical use.

K/3 transport Medium:

growth medium K/1 without Fetal Calf Serum (FCS) (see example 1). However, simple physiological saline solutions may also be employed, for example based on phosphoric-boric acid, such as PBS, or based on HEPES (N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid) or MES ([ 2-N-morpholino ] ethanesulfonic acid).

2. Transport of cryo-preserved wound healing dressings

Wound healing dressings that have been cryo-preserved are typically shipped to the clinic on dry ice. However, other forms of transport may be used, provided that transport of the wound healing dressing below-60 ℃ is ensured.

The wound healing dressing that had been cryopreserved was quickly thawed. The freezing medium is then removed and the dressing is rinsed 1 or more times with K/3 transport medium (see above) or other suitable physiological solution, such as Ringer's solution.

3. Therapeutic uses

The dressing is then placed over the wound. When a non-porous carrier material is used, the wound-healing dressing must be properly positioned so that the cells contact the wound surface. The use of such porous carriers which allow keratinocytes to colonize both sides of the carrier means that the wound healing dressing of the invention need not be placed in any particular orientation on the wound to be treated. This treatment can be repeated multiple times depending on the effect of the treatment.

Example 6: genetic characterization of keratinocytes KC-BI-1(DSM ACC2514)

By adopting the method, KC-BI-1 cells are cultured for several generations. Genetic analysis of the cells at passage 4, 13, and 121 investigated the length polymorphisms of 15 different loci (CSF1PO, D13S317, D16S539, D18S51, D21S11, D3S1358, D5S818, D7S820, D8S1179, FGA, PentaD, Penta E, TH01, TPOX and vWA). It is analyzed using methods known in the art. For this purpose, the corresponding alleles were amplified using a test kit (PowerPlex * 16 System) from Messrs Promega (Mannheim, Germany) which identifies paternal origin according to the instructions provided by the manufacturer. Alleles were identified by determining fragment length (standard length ILS 600 is part of the kit described above). The corresponding tables contain the allele frequency data of the population.

All allelic analyses at all loci were consistent for all cell generations analyzed. This data thus enables KC-BI-1 cells to be genetically classified. The classification probability was determined to be > 99.999% from the allele frequency.

Determination of DNA length polymorphism

Marking	Donor	KC-BI-1 generation 4	KC-BI-1 generation 13	KC-BI-1 generation 121
					CSF1PO	1112	1112	1112	1112
D13S317	813	813	813	813
					D16S539	1113	1113	1113	1113
D18S51	1317	1317	1317	1317
					D21S11	2929	2929	2929	2929
D3S1358	1515	1515	1515	1515
					D5S818	911	911	911	911
D7S820	99	99	99	99
					D8S1179	1014	1014	1014	1014
FGA	2326	2326	2326	2326
					Penta D	1112	1112	1112	1112
Penta E	1012	1012	1012	1012
					TH01	89,3	89,3	89,3	89,3
TPOX	810	810	810	810
					vWA	1618	1618	1618	1618

Reference to the literature

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H. rle-Bachor C; boukamp P (1993) telomerase activity in keratinocytes of the epidermis of human skin regenerating the basal layer and skin of immortal cancer origin, PNAS 93; 6476-6481

Falanga V et al (1998): rapid healing of venous ulcers and clinically ineffective treatment of human skin equivalents cultured with allogeneic tissue transplantation, dermatological archive (Archives of Dermatology), 134, 293-

Harris PA; di frannesco F; barisoni D; leigh IM; navasaria (1999): use of hyaluronic acid and cultured autologous keratinocytes and fibroblasts in large area burns, lancets (Lancet), 353, 35-36

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Lam PK; chan ESY; edward WH et al (1999): a novel composition-the development and evaluation of Laserskin grafts, journal of traumatology, Trauma, Infection and risk treatment (J.of Trauma: Injury, Infection and clinical Care), 47; 918-922

Lang E; sch die BM; eickhoff U; hohl HP; kramer, M D; Maier-Reif K (1996): rapid homogenization of epithelial integrin expression following human keratinocyte allograft, Journal of dermatological Dermatology, 107, 423-427

Leigh IM; navsaria H; purkis PE; McKay I (1991): clinical practice and biological Effect study of keratinocyte transplantation, annual report of medical blood sample (Annals of the academic of Medicine), 20(4)

Lindgren C; marcusson JA; toftgard R (1998): treatment of venous leg ulcers with cryopreserved cultured allograft keratinocytes: one prospective prospectively controlled study, the British Journal of Dermatology, 139(2)271-5.

Maier K (1993): epidermal transplantation in vitro culture-chancn and Risiken, quintesenz 3: 289-304

Phillips TJ; GilchestBA (1989): culture of allogeneic tissue transplanted keratinocyte graft in treatment of wound healing: predictor, Journal of dermatology, Surgery and Oncology (Journal of dermatology Surgery and Oncology), 15(11)

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Sch*nfeld M；Moll I；Maier K；Jung EG(1993)：Keratinozyten aus derZellkultur zur Therapie von Hautdefekten.Hautarzt，44：281-289

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Claims (25)

1. A keratinocyte cell that is incapable of immortalization but capable of proliferation for at least 150 passages by in vitro cell culture.

2. The keratinocyte cell of claim 1, isolated from an epidermal portion of a foreskin.

3. Keratinocytes according to claim 1, characterized in that they are cells derived from KC-BI-1(DSMAC 2514) cultures, or keratinocytes derived therefrom.

4. The keratinocyte cell of claims 1-3, wherein the keratinocyte cell

a. In the absence of fetal bovine serum and/or

b. In the absence of feeder cells and/or

c. Cannot replicate in the absence of Epidermal Growth Factor (EGF).

5. The keratinocyte cell of claims 1-4, wherein said keratinocyte cell has little or no telomerase activity, particularly when compared to immortalized keratinocyte cells, particularly when compared to a HaCaT cell line.

6. Keratinocyte cell according to claims 1 to 5, characterized in that said keratinocyte is capable of replicating for at least 200 passages by means of in vitro cell culture.

7. Keratinocyte cell according to claims 1-6, characterized in that said keratinocyte is capable of replicating for at least 250 passages by means of in vitro cell culture.

8. Keratinocyte cell according to claims 1-7, characterized in that said keratinocyte is capable of replicating for at least 300 passages by means of in vitro cell culture.

9. A product consisting of a carrier coated with keratinocytes according to any one of claims 1 to 8,

a. the carrier is partially covered with keratinocytes; or

b. The carrier is completely covered with keratinocytes.

10. The product of claim 9, wherein the carrier is a biocompatible carrier material useful for preparing pharmaceutical compositions.

11. The product of claim 10, characterized in that the carrier material is a hydrophilic or hydrophobic biodegradable film.

12. The product of claim 10 or 11, characterized in that the carrier is an esterified hyaluronic acid polymer, preferably a porous polymer membrane having specified geometrical characteristics,

a. the polymer film has a thickness of 10 to 500 mu m and is provided with pores having a pore diameter of 10 to 1000 mu m, the pores having a definite, uniform size and being arranged in an ordered arrangement at a constant interval of 50 to 1000 mu m from each other.

13. The product of claim 10 or 11, characterized in that the carrier material is a polyester, polycarbonate, polyanhydride, polyorthoester, polyglycopeptide, polyetherester, polyamino acid or polyphosphazene,

a. in particular: poly (L-lactide), poly (D, L-lactide), poly (L-lactide-co-D, L-lactide), poly (glycolide), poly (L-lactide-co-trimethylene-carbonate) or poly (dioxane),

b. wherein said polymer is porous or

c. Is non-porous.

14. Method for the cryopreservation of keratinocytes as claimed in claims 1 to 8 or of products as claimed in claims 9 to 13, wherein said keratinocytes or products are cryopreserved at a temperature of from-20 ℃ to-196 ℃, preferably from-60 ℃ to-80 ℃.

15. The keratinocyte cell of claims 1-8 or the keratinocyte cell and carrier-containing product of claims 9-13, which has been treated with the method of claim 14.

16. Keratinocytes as claimed in claims 1 to 8 and 15 or products as claimed in claims 10 to 13 and 15 for medical use.

17. Use of keratinocytes as claimed in claims 1 to 8 and 15 or of products as claimed in claims 10 to 13 and 15 for the treatment of wounds.

18. Use according to claim 17, wherein the wound is preferably a burn and/or ulcer.

19. The use of claim 17, wherein the wound is preferably a second degree burn.

20. Use according to claim 17, said wound being preferably a difficult-to-heal chronic ulcer of the lower extremities Ulcus cruris, preferably Ulcus cruris venosum.

21. The use according to claim 17, wherein the wound is preferably an ulcer caused by diabetes.

22. Use according to claim 17, wherein the wound is preferably a decubitus ulcer.

23. Use according to claims 16-22, as a supplement to or in combination with one or more other substances effective in the treatment of wounds.

24. The use according to claim 23, wherein the other substance is a hydrocolloid dressing.

25. Use according to claim 23, wherein the other substance is an antimicrobial substance, e.g.

(a) An antibiotic.