DE3829183A1 - FEEDER-LAYER FROM HUMAN BINDING TISSUE CELLS, METHOD FOR THE PRODUCTION THEREOF AND METHOD FOR THE PROPOSITION OF HUMAN EPITHELIC CELLS AND THE USE THEREOF FOR THE RESTITUTION OF THE SKIN - Google Patents

Abstract

Method of producing a feeder layer based on normal diploid human connective tissue cells, and also the use of this feeder layer as a substrate for the in vitro propagation of normal human epithelial cells, particularly keratinocytes of interfollicular or intrafollicular origin, and also of acinar and tubular cells of the eccrine sweat gland or sebaceous gland cells, and in addition the use of the epithelial cells propagated in this way for skin restitution. The feeder layer is characterized by having a density of 4000-5000 cells per square centimetre.

Description

Feeder layers from human connective tissue cells, Process for its preparation and process for Multiplication of human epithelial cells and their use for restitution of the skin

The present invention relates to the manufacture of a Feeder layers from normal diploid human connective tissue cells, especially fibroblasts, which initially isolated from healthy human skin connective tissue and can then be multiplied in vitro, and the use of the feeder layer for in vitro propagation of normal human keratinocytes, sweat glands and Sebaceous cells, especially keratinocytes, which then alone or in combination with in collagen gels incorporated untreated human fibroblasts for restitution of the skin, for example after Burns or other skin defects, be used.

It is known, for example from the article G.G. Gallico et al., N. Engl. J. Med. 311, 448-451 (1984) that interfollicular human keratinocytes in propagated in vitro and then to cover Burns can be used. An essential one The disadvantage of this method is that treating patients additionally surgically healthy skin must be taken to source material for the In Vitro reproduction of the body's own keratinocytes.  

The amount of skin material to be removed from the patient according to J.G. Rheinwald and H. Green, Cell, 6, p 331-344 (1975) and DE-PS 26 51 685 be reduced if the number of keratinocytes Primary cultures a feeder layer consisting of 3T3- Cells of the mouse that is taken to help. However, since the 3T3 cells are of animal origin and also certain characteristics characteristic of transformed cells own, is their use as a feeder layer for the In vitro multiplication of human cells, which then be transplanted back to the patient, not harmless.

Furthermore, from the literature A. Limat and F. K. Noser, J. Invest. Dermat. 87, 485-488 (1986) that intrafollicular Keratinocytes from human hair follicles, for example, plucked cells of the outer root sheath human anagen hair of any anatomical origin, can be multiplied in vitro if the 3T3 feeder layer mentioned above is used.

It has also been tried, for example by H. P. Baden et al., J. Invest. Dermat. 76, 53-55 (1981) normal human fibroblasts as feeder layers for the To use proliferation of human epithelial cells however, the growth of the epithelial cells was there after that method used unsatisfactory.

The task was therefore to develop a process for the In Vitro propagation of normal human keratinocytes, Sweat or sebum cells are available too which is at least as reproducible and propagation results as good as the procedure with  3T3 feeder layers, but without their potential Disadvantages in immunological and possibly also to be infectious.

It has now been found that a feeder layer is starting from normal human diploid connective tissue cells, especially normal human fibroblasts, which are prevented from multiplying, excellent for that In Vitro Multiplication of Normal Human Epithelial Cells the skin, especially keratinocytes interfollicular or intrafollicular origin, as well as acinar and tabular cells of the eccrine sweat gland or sebaceous cells is appropriate if the feeder layer cells in the density according to the invention from 4000 to 5000 Cells / cm² are plated out.

The invention therefore relates to a method for Production of a feeder layer from human Connective tissue cells, where you have normal diploids human connective tissue cells treated so that they then prevented from multiplying, then this plated in a suitable nutrient medium and in there cultivated in a known manner until it has a flat, assume epithelioid form, characterized in that the connective tissue cells in a density of 4000 to 5000 cells / cm², preferably 4500 cells / cm², plated out.

For example, are suitable as feeder layer cells human fibroblasts autologous or heterologous Origin of the differentiation stages II-VI according to K. Bayreuther et al., Proc. Natl. Acad. Sci. USA 85, 1-5 (1988), with fibroblasts of the differentiation stages IV-VI are preferred.  

The prevention of the proliferation of connective tissue cells takes place either by treating connective tissue cell cultures with a cell density of about 6000-9000 Cells / cm², preferably 8000 cells / cm², with 6-12 Micrograms / ml, preferably 8 micrograms / ml, mitomycin C for 4-8 hours at 30 to 37 degrees Celsius or by irradiation of trypsinized connective tissue cells with a cell density of about 10⁵ to 10⁷ cells / ml, preferably 10⁶ cells / ml, with a dose of 5000 -10 000 cGy (Zentigray), preferably 7000 cGy, by a ionizing radiation source.

Starting from explanted skin connective tissue from healthy people, connective tissue cell cultures are prepared using known methods, for example according to WS Sly et al. Methods in Enzymology, Vol. LVIII-Cell Culture, pages 444-450, Academic Press, New York (1979). Subsequently, connective tissue cell cultures with a cell density of 6000-9000 cells / cm², preferably 8000 cells / cm², in DMEM (Dulbecco's modified Eagle's medium) containing 10 volume percent FCS (fetal calf serum) and 6- 12 micrograms / ml, preferably 8 micrograms / ml ml contains mitomycin C, incubated for 4 to 8 hours, preferably 6 hours, at 37 degrees Celsius. The fibroblasts are then washed 4 times with PBS (phosphate buffered saline), removed with the aid of a solution of 0.05% by weight of trypsin and 0.02% by weight of EDTA in Ca ²⁺ and Mg ²⁺ -free PBS, centrifuged off and, according to the invention, at a density of about 4000-5000, preferably 4500 cells / cm 2, plated under incubation conditions customary for cell cultures in DMEM which contains 10 volume percent FCS.

According to a second embodiment of the invention Preconfluent cultivated connective tissue cell cultures are used as above for the first method described trypsinized. Then the cells in a density of about 10⁵-10⁷ cell / ml, preferred 10⁶ cells / ml, under conditions customary for cell cultures suspended in DMEM containing 10 volume percent FCS contains. The portions, preferably filled into tubes this cell suspension of 2 ml with a Single dose of ionizing radiation of 5000-10 000 cGy, preferably 7000 cGy, irradiated.

The connective tissue cells treated or irradiated with mitomycin C. are for use as feeder layers both freshly sown and after Storage under normal incubation conditions for cell cultures at 30 to 37 degrees Celsius, preferably 37 degrees Celsius, up to 4 months, especially 1-2 months, after sowing or after cryopreservation via any Periods ideally suited. Cryopreservation of the feeder layer cells is done as follows: To cryopreserve irradiated cells the medium containing the feeder layer cells before the irradiation 5 to 10 volume percent DMSO (dimethyl sulfoxide) or 5 to 10 volume percent glycerin added. The feeder layer cells are immediately after the Irradiation frozen according to the usual methodology. Mitomycin C-treated feeder-layer cells are 14-24 In mitomycin for hours, especially 20 hours C-free, containing 10 volume percent FCS DMEM culture medium kept, treated with trypsin, centrifuged and under usual for cell cultures Conditions in a 5 to 10 volume percent DMSO or 5 up to 10 percent by volume  Medium containing glycerin and then resuspended frozen according to the usual methodology.

The invention further relates to a feeder layer obtained from normal diploid human connective tissue cells, which has a cell density of 4000 to 5000 Has cells / cm², especially one that by the procedure described above was obtained.

Irradiated connective tissue cells show 24 hours after Irradiation mostly takes the form of an elongated one Spindle on. The cell area increases in the subsequent Incubation period is still increasing considerably. After 3-4 days the irradiated connective tissue cells predominantly have one flattened-epitheloid shape. Mitomycin C treated Connective tissue cells show 24 hours after treatment predominantly flattened-epitheloid form. The Cell area increases during the subsequent Incubation period is still increasing considerably.

So that the feeder layer cells perform their feeder function they have to be differentiated can spread fully accordingly; at the same time enough space for the multiplying skin epithelial cells left over. The feeder layer capacity of the connective tissue cells is determined by the anatomical origin and the number The population doubled through cannot be measured influenced.

In contrast to 3T3 feeder layers is in use of feeder layers from human connective tissue cells none during the cultivation of keratinocytes Renewal of the feeder layer necessary.  

It has also been found that, in contrast to the 3T3 feeder layers, in the feeder according to the invention Layering the adherence to the culture surface is optimal.

Those made by the method described above Connective tissue feeder layer cells are therefore very much easier to handle than the 3T3 feeder layer cells, which while in use as a feeder layer tend to get out of the culture surface prematurely peel off or even frequently for no apparent reason degenerate.

The invention further relates to a method for In Vitro Multiplication of Normal Human Keratinocytes the skin, acinar and tubular cells of the ecrine sweat gland as well as of sebaceous cells, whereby you start with a feeder layer based on normal diploid human connective tissue cells after the methods described above in a density of produces about 4000 to 5000 cells / cm² and then then the keratinocytes and / or sweat glands and / or Sows sebaceous cells.

Particularly good results are obtained if the keratinocytes, Sweat or sebum cells in one Density from 50 to 10,000, especially 500 to 4000 Cells / cm², are sown.

Of the keratinocytes that can be used are the interfollicular ones or intrafollicular keratinocytes are preferred.

The above proved to be very particularly suitable described feeder layer for the culture of keratinocytes from the outer root sheath of hair follicles more human Anagen hair.  

Depending on the age of the people, 30 (for young People) up to 60 (in older people) anagen hair plucked. To avoid contamination by Keratinocytes or epidermis or the hair bulb at the level of the sebaceous gland the distal part and proximally removed portions of the hair bulb and discarded. From the remaining hair follicle piece, the Outer root sheath keratinocytes by treatment with trypsin, as in A. Limat and F. K. Noser, J. Invest. Dermat. 87, 485-488 (1986). It about 5000-10,000 individual keratinocytes / Get hair follicles. For the production of primary cultures these are sown in the above-mentioned publication A. Limat and F. K. Noser described FAD nutrient medium at a density of about 500-4000 cells / cm² on the feeder layer described above, obtained from normal diploid human connective tissue cells of the Density of about 4000-5000 cells / cm².

The primary keratinocyte cultures obtained in this way can as well as subcultures based on it, for which is either the FAD culture medium or the MCDB 153- Culture medium (Clonetics Corporation, San Diego, USA) was used directly for the back transplant to the Patients are used.

In the method according to the invention, for example, grew intrafollicular keratinocytes, within 2 to 3 weeks to confluence, displacing the Feeder layer cells that detached from the base and eliminated quantitatively when changing the medium could become. The growth rate of primary intrafollicular Keratinocyte cultures were about 4 to 8% (n = 6), the number of population doubles was increased 9.6 determined within 20 days. Preconfluent  Cultures can also be Layer cells by treatment with 0.02 percent by weight aqueous EDTA solution are freed.

The keratinocyte cultures, which are about 2 to 7 cell layers are thick, can be used for back transplantation Dispase II (neutral protease, available from Boehringer / Mannheim) from the bottom of the culture vessels.

Secondary cultivation is optimal when keratinocytes the primary culture, which is still in the preconfluent stage.

The grown according to the method of the invention Keratinocytes are in terms of the keratin expression pattern identical to keratinocytes in situ and with keratinocytes, which was grown using 3T3 cells were. With regard to morphological criteria, they are according to the invention grown keratinocytes identical to those grown using 3T3 cells; in particular, they have a typical epitheloid shape with a high nucleus / cytoplasm ratio.

The excellent results in normal propagation human keratinocytes according to the invention Methods are state of the art, for example DE-PS 26 51 685, completely surprising. There was namely found that the 3T3 feeder layer for propagation human epidermal cells with a density of about 1.5-5 × 10⁴ cells / cm² is to be sown while one Sowing density of the epidermal cells of about 50-5000 Cells / cm² is appropriate. Besides are after that Publication of 3T3 cells versus human  diploid fibroblasts regarding their use as Preferred feeder layer.

It was also found that the growth of Keratinocytes drastically decreased when the number of 3T3 feeder layer cells to values below 20,000 Cells per cm² is reduced. In contrast, the the inventive method to determine that just at the much smaller number of 4500 according to the invention Cells per cm² an optimal multiplication of the keratinocytes is guaranteed. This is as above mentioned, due to the fact that the much larger feeder cells according to the invention a much higher Show space requirements as corresponding 3T3 cells.

In summary, for the method according to the invention for the proliferation of keratinocytes, sweat glands or Sebaceous cells have the following advantages over the known ones Propagation with 3T3 feeder layers are called:

• 1. The use of normal human diploid Connective tissue cells in contrast to aneuploid, poorly defined and transformed, so regarding normal growth and metabolism Different cells, mouse cell lines.
• 2. The possibility of being heterologous and for special Issues also include autologous connective tissue cells use.
• 3. The ability to anatomically connect the connective tissue cells defined places to use.  
• 4. The better reproducibility for the in vitro Proliferation of normal skin epithelial cells due to the fact that human connective tissue Feeder layers are easier to handle.
• 5. The ability to layer human connective tissue feeders for a period of at least 4 weeks without noticeable loss of feeder effectiveness at 37 Degrees Celsius below usual for cell cultures Keep incubation conditions.
• 6. The ability to feed human connective tissue Layer cells indefinitely in frozen Keep condition.
• 7. The reduction of potential immunological and also infectious (viral) hazards in the Use based on the feeder layer increased human keratinocytes, for restitution of the skin.

The invention therefore also relates to the use of keratinocytes increased by the above method, Sweat glands or sebum cells for restitution the skin, for example after burns or if there are other large skin defects.  

During the in vitro cultivation of keratinocytes, which preferably come from the patient himself (autologous transplantation), the time required, the Skin defects temporarily covered and for that subsequent transplant prepared (avoidance of Dehydration, stimulation of granulation, infection control). About 2 to 8 are used for the transplant Time slots of thick keratinocyte cultures with Dispase II from Detached from the bottom of the culture vessels, with Haemoclips in the Culture vessel with suitable dressing material, for example Sofratulle, pinned and then on the cleaned and from excessive granulation tissue released wound areas and possibly sewn on. The further treatment follows the general Guidelines for skin grafts.

The following examples are intended to illustrate the invention explain without the invention to these examples restrict.

Examples Example 1 Production of a feeder layer starting from of normal human fibroblasts

Starting from explanted healthy human skin connective tissue or from diploid human fibroblasts Cell strains obtained from Human Genetic Mutant Cell Repository, Camden, New York, were made famous for Methods, such as in W. S. Sly et al., Methods in Enzymology, Vol LVII - Cell Culture, pages 444-450, Academic Press, New York (1979), Fibroblast cultures are grown and according to one of the following described method A or B treated further.

Procedure A

The fibroblast cultures obtained as stated above are at a density of 8 × 10³ cells / cm² in DMEM (Dulbecco's modified Eagle's medium), which contains 10 volume percent FCS (fetal calf serum) and 8 micrograms / ml mitomycin C, for 6 hours incubated for long at 37 degrees Celsius. The fibroblasts which are prevented from growing by this treatment are then washed 4 times with PBS (phosphate buffered saline), detached with the aid of a Ca ²⁺ and Mg ²⁺ -free PBS containing 0.05% by weight of trypsin and 0.02% by weight of EDTA, and then centrifuged off.

Procedure B

The preconfluent fibroblast cultures obtained as above are detached with the aid of a Ca ²⁺ and Mg ²⁺ -free PBS containing 0.05% by weight of trypsin and 0.02% by weight of EDTA, centrifuged off and then at a density of 10⁶ cells / ml in DMEM , which contains 10 volume percent FCS, suspended. Portions of 2 ml of this cell suspension filled into tubes are irradiated with a single dose of 7000 cGy (radiation device and conditions: Dermopan 2 (from Siemens), 50 kilovolts, 1.0 mm aluminum filter, distance 5 cm).

Fibroblasts treated according to procedure A or B. are excellent for use as feeder layer cells suitable and are used for this purpose in a density from 4000 to 5000 cells / cm² in DMEM, which is 10 volume percent FCS contains plated.

The following diploid human shown in Table 1 Cell strains were described in the above Way used as feeder layer:

Table 1

Example 2 Cryopreservation according to example 1 obtained feeder layer cells

The obtained according to method A (Example 1) with Feeder-layer cells treated with mitomycin C turn 24 Mitomycin C-free 10 volume percent FCS for hours containing DMEM, then typed, centrifuged, then in 10 volume percent dimethyl sulfoxide containing medium and then resuspended frozen.

To get to B (Example 1) irradiated cells cryopreserve, the one containing the feeder layer cells Medium before irradiation 10 percent by volume Dimethyl sulfoxide or glycerin added. Then be the feeder layer cells irradiated and immediately after radiation frozen.  

The freezing process takes place every 24 hours Cool to -80 degrees Celsius and then store in liquid nitrogen.

The cryopreserved in the manner described above Feeder layer cells are over any period of time durable.

Example 3 In vitro propagation from normal human Keratinocytes

A. Keratinocytes of the outer root sheath of plucked human hair follicles are, as in A. Limat and FK Noser, J. Invest. Dermatol. 87, 485-488 (1986) described: 40 hair follicles are in DMEM, which is buffered with 25 mmol HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 400 U / ml penicillin and 400 micrograms / ml streptomycin contains, suspended. The hair follicles of hair in the anagen (growth) phase are carefully selected using a microscope. For certain research purposes, the upper part of the hair can be cut to the level of the sebaceous gland and the hair bulb in order to avoid contamination by keratinocytes of the epidermis or the hair bulb. After 2 more rinses with DMEM, the remaining parts of the hair follicles are transferred to a petri dish and the liquid is suctioned off. The follicles are then 10 with 0.15 ml of a PBS containing 0.1% by weight of trypsin and 0.02% by weight of EDTA, which is free of Ca ²⁺ and Mg ²⁺ ions and has a pH of 7.2 Incubated for minutes at 37 degrees Celsius. Then a vigorous pipetting of the follicle suspension in DMEM mixed with 10 volume percent fetal calf serum gives a suspension which mainly consists of individual cells.

After centrifuging at about 2000 m / s² for 10 minutes the keratinocytes are back in the culture medium suspended. The keratinocytes obtained in this way from the outer root sheath more human Hair follicles are in a density of 1 × 10³ Cells / cm² on a feeder layer, obtained from normal diploid human fibroblasts, which was produced according to Example 1 and a density of 4.5 × 10³ cells / cm² in a 10 cm culture dish sown in FAD medium. In the course of 2-3 The keratinocytes grow to confluence for weeks and displace the feeder layer cells, which detach from the pad and when changing the Culture medium are eliminated quantitatively. The so obtained primary cultures of keratinocytes or based on this subcultures (nutrient medium FAD or MCDB 153 (Clonetics Corporation, San Diego, USA)) of the area required in each case can be used directly for the transplant.

B. Keratinocytes of the juvenile foreskin and other tissue samples from children and adults are isolated by 1.5 hours of treatment with 0.25 percent trypsin in Ca ²⁺ and Mg ²⁺ -free PBS at 37 degrees Celsius and grown in the same way, as described in example 3, paragraph A.

Example 4 Use of the keratinocyte cultures for Restitution of the skin

The manufactured according to Example 3 in the required size Keratinocyte cultures (primary or subcultures) which about 2-8 cell layers are known to be known Procedure with Dispase II (Boehringer Mannheim) from the ground the culture vessels detached, with haemoclips in the culture vessel attached to the sofa truss, then to the cleaned, freed from excessive granulation tissue Wound surfaces transferred and then either sewn on (for example in the case of burns or after larger excisions like cutaneous hamartomas or Melanomas) or by means of additional layers of sofa and Compression bandage (for example in the case of ulcers cruris) fixed. The further treatment takes place after the respective general guidelines for skin grafts.

Claims (13)

1. A method for producing a feeder layer from human connective tissue cells, in which normal diploid human connective tissue cells are treated in such a way that they are subsequently prevented from growing, then plated out in a suitable nutrient medium and cultivated there in a manner known per se until they are assume a flat, epitheloid shape, characterized in that the connective tissue cells are plated at a density of 4000 to 5000 cells / cm². 2. The method according to claim 1, characterized in that that the connective tissue cells in a density of 4500 Cells / cm² are plated out. 3. The method according to claim 1 or 2, characterized in that as connective tissue cells human Fibroplasts of autologous or heterologous origin Differentiation stages II-VI according to K. Bayreuther, Proc. Natl. Acad. Sci. USA 85, 1-5 (1988), be used. 4. The method according to any one of claims 1 to 3, characterized characterized in that the connective tissue cells by Treatment with 6-12 micrograms / ml mitomycin C during 4-8 hours at 30-37 degrees Celsius are prevented from multiplying. 5. The method according to any one of claims 1 to 3, characterized characterized in that the connective tissue cells by Irradiation with a dose of 5000-10 000 centigray through an ionizing radiation source are prevented from multiplying.   6. The method according to any one of claims 1 to 5, characterized characterized in that the connective tissue cells in one Nutrient medium consisting of 10 volume percent FCS (Fetal calf serum) spiked DMEM (Dulbecco's modified Eagle's medium). 7. Feeder layers obtained by a method according to a of claims 1 to 6. 8. Feeder layers obtained from normal diploids human connective tissue cells, characterized in that its cell density is 4000 to 5000 cells / cm² is. 9. Procedure for In Vitro Multiplication of Normal human keratinocytes of the skin, acinar and tubular cells of the eccrine sweat gland as well as of Sebaceous gland cells, characterized in that one first a feeder layer based on normal diploid human connective tissue cells after a Manufactures method according to claims 1 to 6 and then the keratinocytes and / or Sows sweat glands and / or sebum cells. 10. The method according to claim 9, characterized in that the keratinocytes, sweat gland or sebaceous cells in a density of 50 to 10,000, in particular 500-4000 cells / cm² are sown. 11. The method according to claim 9 or 10, characterized in that keratinocytes are interfollicular or intrafollicular origin.   12. The method according to any one of claims 9 to 11, characterized characterized that keratinocytes from the outer Root sheath of hair follicles of human anagen hair used. 13. Use of the propagated according to one of claims 9 to 12 Keratinocytes, sweat gland or sebum cells for restitution of the skin.