KR20010072553A - A Living Chimeric Skin Replacement - Google Patents

Abstract

The present invention includes skin substitutes to enhance wound healing. According to one embodiment, this skin replacement is chimeric, comprising obtaining self-recognized epithelial cells from a patient and then inoculating the epithelial cells into a biocompatible substrate containing in vitro cultured dimeric epithelial cells. Such live chimeric skin substitutes may be implanted on permanent skin substitutes that have already been implanted at the wound site. In another embodiment, a skin substitute may comprise (a) an internal component comprising a biocompatible dermal structure having a biodegradable or removable backbone as the base material; (b) an intermediate component comprising epithelial cells; And (c) an external component comprising epithelial cells cultured in vitro on a dermal construct comprising a dermal addition comprising a biodegradable or removable backbone as a base material. The present invention also encompasses methods of making and implanting the various skin substitutes according to the present invention.

Description

Living Chimeric Skin Replacement

The present invention relates to live chimeric skin substitutes for improving wound healing. More specifically, the compositions and methods of the present invention provide a permanent replacement skin comprising in vitro cultured two-factor epithelial cells that can be used to fill a wound, as well as self-recognized epithelial cells that can be implanted immediately. The present invention provides a source of cells that can completely cover a wound and also provides the patient's own keratinocytes.

Many skin wounds that cause breaks, burns, punctures and skin ulcers often require clinical treatment. However, conventional treatments for such wounds may yield unsatisfactory results. In the United States, about 10,000 people a year die from skin injuries from severe burns.

Life-threatening severe burns occur quite frequently during military service and other social activities. Thus, there is a clinical need to better survive and protect patients with severe skin wounds such as burns.

The skin is composed of epidermal and dermal tissue. The epidermis is composed of acid and dead layers. The basal layer and the superficial layer, also called the maldermal layer, function to make the outermost dead layer, the stratum corneum. The epidermis grows from the basal layer of the core. Between these columnar monolayers are scattered keratinocytes, pigment-producing cells called melanocytes, and Langerhans cells, which are made from the bone marrow and play an important role in the immune function of the skin. The cells proliferate at the bottom of the basal layer and the pole layer (or the superstrate layer) and push out continuously towards the surface.

Epidermal cells migrate toward the skin surface, becoming thinner, dehydrated, cured and keratinized (aka, differentiation process). The upper layer consists of several cell layers substantially. Cells migrate from the pore layer to granule layers, typically two to three cells thick. Over time, the cells migrate from the granular layer to the transparent layer, a region where keratinization occurs, where the disulfide bridges form between cysteine residues to stabilize the membrane structure. Fully formed, flattened and hardened epidermal cells enter the stratum corneum.

The dermis consists primarily of supportive connective tissue, including fibrous tissue containing blood vessels and nerve branches. It also includes fibroblasts that form components of connective tissue such as extracellular matrix proteins (eg, collagen, fibronectin, elastin, etc.). These matrix proteins provide skin strength and flexibility. The dermis is partially but attached to the epidermis by the basement membrane. The skin also contains appendages such as hair follicles and sweat glands. Depending on the severity of the wound, some or all of the above skin components may be damaged or lost.

Currently, in the United States, patients with extensive burns are treated with surgical removal of the wound or by autografting unburned skin obtained from the patient. Techniques such as mesh tissue transplantation are used to properly cover and promptly treat the wound. This technique cuts the slit into a skin graft that spreads over the wound to cover the wound. However, it is difficult to obtain healthy skin, so it may be necessary to reacquire it at the supply site. However, this can lead to complex problems such as blood loss, infection concerns and delayed treatment. In other words, for the treatment of patients suffering from ectopic burns, there is a serious problem that at least several months must pass before the autologous implant can be regained enough to completely cover the wound from the supply site. Therefore, the cuticles are usually meshed and stretched to cover a wide range of wounds in order to reduce the duration of treatment. However, the method of stretching after such a mesh treatment has a problem that the wound remains permanently and furthermore, an undesirable mesh form is left in a wide and excessive amount in the cured skin. In addition, the method of reconstructing the skin by a surgical operation also has a problem that takes several years.

If only a small amount of unburned skin can be obtained, the two-dimensional graft of the body skin (frozen or fresh) may be used as a biological standard dressing to dislodge the cutout. Currently used (Atnip and Burke, 1983, Curr, Prob. Surg. 20: 623-86; Furut and Levin, 1984, Arch. Surg. 119: 312-22; Hansburg, 1987, In Boswick Jay, The Art and Science of Burn Care, Rockville, MD: Aspen Press, 57-63).

If autologous skin cannot be obtained, it is better to transplant a fresh body skin to cover the cut out, but there is a problem that the supply of fresh skin is limited. Two-factor transplantation of carcass skin allows longer lived patients whose immunity is significantly suppressed to the extent that burns are severe, but has the disadvantage of causing an immune rejection response within a few weeks after transplantation (Ninemann et al., 1978, Transplantation 25: 69-72). If rejection of the factor transplanted skin occurs, the procedure should be repeated. Such rejection reactions can involve infection of bacterial colonies and wounds, which later prevents transplantation of either two-factor skin or self-recognized skin. In addition, later transplantation may further facilitate rejection as a result of sensitization of the immune system. Treating patients with extensive burns requires timely transplantation before life-threatening infections and other complex problems occur, which is a battle against time.

Since 1983 reported the use of cultured two-factor implants (Hefton et al., Lancet 11: 428, 1983), attempts have been made for clinical use. Some early scholars argued that cultured implants have long survival and immune benefits. However, these studies have been performed on wounds of non-uniform thickness, and cannot exclude the possibility that epithelium may have formed from dermal components such as hair follicles and the like. In addition, a recent study (see Philips, T. J. Transplantation 44: 106; 1987) suggested that cultured keratinocyte-forming bifactor implants could not survive permanently. HLA I assay (Giellen et al., Dermatologia 175: 166 (1987)), DNA Muggerprint method (Druka et al., Burns 15: 303 (1989)) and Y-chromosome assay (Burt et al., Br. Med. J. 298: 915 (1989); Brain et al., Br. Med. J. 298: 917 (1989) are proof documents for a bifactor implanted replacement by receptor keratinocytes.]

Another approach to obtaining more implantable tissue is to obtain intact skin fragments from the patient and allow epithelial cells present in biopsy to be grown in in vitro tissue culture. For example, US Pat. No. 4,016,036 to Green and Rheinwald discloses a technique for continuous culturing of human epidermal cells (keratin forming cells, etc.), wherein primary culture of keratinocytes is performed on the total thickness of human skin. Start with a single cell suspension obtained from a biopsy (chopped and trypsin isolated to separate cells). The cell suspension is transferred to a petri dish containing a culture medium containing several growth factors. This cell suspension is poured onto a layer of fibroblasts in rats which prevented proliferation by radiation treatment. To obtain more keratinocytes, secondary cultures are initiated by treating the culture with trypsin and passing the isolated cells from them through several dishes. Keratin forming cell colonies are combined with adjacent colonies to grow until a fusion sheet of keratin forming cells is made.

US Pat. No. 4,304,866 to Green and Keihin discloses a technique for detaching cultured self-recognizing keratinocytes from culture dishes. According to this document, the cultured cells were removed from the culture dish by treating the culture medium with dispase, a proteolytic enzyme that decomposes the protein related to the cell-plastic plate attachment while leaving the protein related to the cell-cell adhesion intact. Remove it. However, it takes about three to four weeks to form a sheet of self-recognized cells suitable for skin transplantation, which has the significant disadvantage that the wound healing is considerably slower.

In addition, long-term success of self-aware cultures has been reported to be less than 50%. One such low success factor is the use of neutral proteolytic enzymes (called dispases) that damage cell surface adhesion receptors to remove cells from culture dishes (Renekanf et al., J. Surgical Res. 62: 228-295 (1996).

Pittel Curro and Scott, Mayo Clin. Proc. 61: 771-777 (1986) discloses a phase 2 system for autografting to a patient with extensive burns. The first phase is to obtain small skin samples and incubate in serum-free medium to obtain keratinocyte cell cultures, and the second phase is to induce differentiation to increase keratinocytes and form sheets of differentiated keratinocytes suitable for transplantation. However, this takes about three weeks. In addition, rather than culturing the cells on the membrane, epithelial sheets must be enzymatically removed and trypsinized using daspase prior to transplantation.

Icinger's US Pat. No. 4,299,819 discloses a burn patient, which separates the epidermis from the dermis, breaks down the epidermis into epidermal cells, and incubates the epidermal cells in the absence of components of the dermis in tissue culture at a pH of about 5.6 to 5.9. It is the first to disclose the treatment method. The epidermal sheet is applied here to the wound site of the burn patient.

Gallico et al., New England Journal of Medicine 311 (7): 448-451 (1984), disclose the implantation of skin substitutes using self-recognized epithelial sheets obtained by culturing small skin-biopsy samples. Wherein the self-recognizing cells grow in vitro and involve a number of manipulations that allow the burn patient to be first given a carcass skin double factor implant or collagen-glycosaminoglycan-siltic sheet. Self-recognized skin takes several weeks to grow and sheets of self-recognized cells must be released from the culture flasks using dispase prior to transplantation. In addition, two factor implants should be removed prior to transplanting a self-aware culture. This technique also has the problem of a decrease in body temperature and blood loss following removal of the two factor implant.

Bell's US Pat. No. 4,485,096 discloses skin transplantation in a mouse model inoculated with epidermal cells already biopsied from the host receptor and containing fibroblasts in the collagen lattice. The implant is cultured in vitro and implanted at the wound site.

There are many reasons for the low success rate of skin grafts. For example, the surface molecules of cells may be modified or damaged by enzymes used to remove freshly produced epithelial sheets from tissue culture plates, thereby adhering to or incubating culture cells. Adhesion to the wound may be affected. It is also the reason that multiple cultured epidermal cells can be changed from the proliferation stage to the differentiation stage. Differentiation of cells involves not expressing a specific cell adhesion molecule. As a result, as described above, the adhesion between cells and the adhesion between cells and the wound site are changed so that the wound cannot be covered.

Clinical experience has shown that pure double factor epithelial implants are poorly tolerated in cases of deep burns, but are primarily due to the host's immune response to foreign tissues. Langerhans cells and epithelial cells present in the skin are known to be targets of the host immune response. However, the immune response generally does not occur immediately and may occur quite late in severe wounds such as burns. The immune response that occurs against Langerhans cells and other cells can be alleviated due to damaged tissue around the wound.

There have been a number of attempts to transplant chimeric skin. WO 94/17179 and US Pat. No. 5,610,007 to Auger et al. Disclose the preparation of chimeric epithelium in vitro. Here, co-cultivation of a 50:50 and 25:75 mixture of rat keratinocytes Balb / c and C3H / HeN is followed by enzymatic separation of cells from the flask surface using dispase. The investigators estimated that chimeric implants formed in about one-half of three to five weeks, the time required to form conventional implantable cell sheets. Similarly, Suzuki et al., Transplantation 59 (9): 1236-1241 (1995), examined the keratinocytes of rat keratinocytes C3H / HeN and Balb / c to test the potential for transplantation of cofactor (self-recognition) and two-factor rat keratinocytes. Disclosed is a technique for co-culture. Two factor cells and self-recognizing cells are also co-cultured in vitro and then separated from the culture dish using dispase.

A mixture of two-factor microdermal grafts and self-recognized microdermal grafts (10: 1) was implanted and placed over the entire thickness of the skin wounds of model rats with BIOBRANE (Lin et al., Burns 20 (1): 30). -35 (1994). Burns 20 (1): 534-538 (1994), such as Stark and Kaiser, reported that in vitro incubation of self-recognized keratinocytes from skin biopsies of patients with deep or partial burns on skin Techniques for implanting as non-fused single cells suspended in fibrin glue after 17 and 25 days are disclosed. For some wounds, fibrin suspensions were used to additionally attach glycerolated bifactorial cutaneous skin implants. The culturing of self-recognizing cells prior to transplantation delays transplantation, thus delaying the healing of wounds.

Most of the methods described above require enzyme treatment to isolate cultured cells, which have a negative effect on cell surface molecules required for cell attachment. This is far from the original purpose of the above methods for providing preferred skin implants.

Finally, US Patent No. 5,693,332 to Hansbrug cultivates keratinocytes on hydrophilic membranes for 1 to about 7 days and then cultures self-recognizing keratinocytes for transplantation by contacting the membrane containing keratinocytes with a wound. A method for shortening the time required for the disclosure is disclosed.

In view of the prior art problems described above, it is believed that a better skin substitute is still needed for the treatment of a patient with damaged skin. An ideal skin substitute can be quickly and reproducibly attached to a wound and prolonged without rejection. It should be attached and easily secured in large quantities.

The present invention relates to skin substitutes for improving wound healing.

Summary of the Invention

DETAILED DESCRIPTION The present invention provides living chimeric skin substitutes, methods for making and using the same, which are necessary for treating patients with skin wounds due to various causes including fractures, skin ulcers and burns. The present invention also provides novel complex skin substitutes for the treatment of wounds.

In the present invention, in transplanting in vitro cultured bifactorial epithelial cells (e.g., keratinocytes) onto a membrane or other biocompatible substrate, the present invention may be performed simultaneously with the transplantation such that the self-recognized epithelial cells may be simultaneously transplanted with the bifactorial cells. Partly based on Applicant's breakthrough finding that adding relatively small amounts of self-recognizing epithelial cells to bifactorial cell-substrate constructs at about the same time improves wound healing more. As self-recognizing cells, keratinocytes are preferred, which are seeded on cultured bifactorial cells just prior to transplantation. Self-recognizing cells can also be inoculated directly to the wound site (preferably on top of the dermal replacement construct), and the two-factor cell-substrate construct can be placed on top of the self-recognizing cells so that the two-factor cells face the wound. .

According to one embodiment, the bifactorial cells are cultured in vitro or frozen on membranes or other biocompatible substrates for easy supply of fresh implants. Bifactorial cells are preferably cultured on biocompatible, biodegradable membranes (reversibly hydrated membranes).

According to another embodiment, self-recognizing cells are added to a two-factor implant at a concentration of about 10 ⁴ per square centimeter of implant material.

According to another embodiment, the chimeric implant can be used with the equivalent of DERMAGRAFT (La Jolla Advanced Tissue Science, Calif.) In treating the full thickness of a deep wound. Dermagraft is a living dermal replacement consisting of three-dimensional stromal tissue that comprises connective tissue proteins naturally secreted by stromal cells and attached stromal cells and substantially surrounds the biocompatible frame or backbone. Details of dermagraft are described in US Pat. No. 4,963,489, which is incorporated by reference herein.

According to another embodiment, a complex skin substitute is used in the method of the present invention. That is, a biocompatible dermal construct (e.g., Dermagraft) with a biodegradable or removable skeleton as a base material is placed over the wound, and self-recognized cells obtained from the patient are placed on top of the dermal construct (the dermal construct is vascularized and Concurrent or immediately after vascularization), including bifactorial epithelial cells cultured on dermal components (including biocompatible dermal constructs in the biodegradable or removable skeleton as a base material with transitional coverings such as biobrain membranes) An external component is placed on top of the autologous cell. According to another embodiment of this composite skin substitute, dimorphic cells, self-recognizing cells or combinations thereof are used in the central or external component of the composite replacement.

According to another embodiment, two-factor epithelial cells and self-recognized epithelial cells are inoculated simultaneously into the substrate and incubated for sufficient time for the cells to adhere to the substrate and then transplanted into the wound site so that the cells face the wound.

Detailed description of the invention

The present invention relates to live chimeric skin substitutes. Dermal substitutes of the invention include chimeric constructs that include bifactorial cells and self-recognizing epithelial cells. Bifactorial epithelial cells are cultured in vitro on substrates such as biocompatible membranes, dermal constructs, glues or gels and in most cases are stored frozen. After thawing the bifactorial cell-substrate construct (if frozen), it is placed on the wound site with self-recognized epithelial cells (e.g., keratin-forming cells), mechanically crushed skin or microdermal pieces (where self-recognition). Cells are preferably biopsied immediately prior to transplantation). If the wound is deep, the epithelial replacement can be implanted as a permanent dermal replacement if necessary.

The present invention provides an improved skin substitute that accelerates wound healing by applying a skin covering of bifactorial cells cultured in vitro on substrates such as membranes or dermal structures along with healthy self-recognized epithelial cells to the wound site. The ratio of autologous cells to dimorphic cells may range from 1: 5 to 1:50. Bifactorial cells serve as a covering before an immune response occurs at the wound site. However, self-recognized epithelial cells have advantages both physiologically and immunologically in relation to the wound site and eventually replace the two-factor cells. The amount of self-recognized cells is the amount that the wound can be healed by the self-recognized cell sheath before a significant immune response occurs at the wound site. Therefore, the present invention has the advantage of providing a permanent skin implant that can be used on the fly, whereby a severe immune rejection reaction does not occur by adding self-recognizing cells.

According to one embodiment, the two factor transplanted cells obtained from the body skin are inoculated into host self-recognized cells and then transplanted to the wound site. The self-recognizing cells multiply, and these multiplying self-recognizing cells continue to grow and gradually replace the two-factor tissue as the host's immune response to body tissues occurs.

Bifactorial epithelial cells may be cultured in vitro to allow for immediate transplantation. Bifactorial epithelial cells are cultured on biocompatible, preferably biodegradable substrates.

Live chimeric skin substitutes can be prepared as follows (this method of preparation is only one example and is not limited thereto).

(a) placing bifactorial epithelial cells on a biodegradable, biocompatible substrate to grow to about 25-90% confluence;

(b) frozen storage of the bifactorial cells and substrate until use;

(c) thaw immediately prior to transplantation;

(d) obtaining self-recognized epithelial cells and inoculating them onto the bifactorial cells at a concentration of about 10 ⁴ / cm ² and transplanting them to the wound site;

(e) The chimeric skin implant is covered with a dressing that can be appropriately fixed to prevent mechanical forces or infection from the outside.

(Wherein (b) and (c) are optional steps)

For deep wounds, dermal substitutes are additionally used at the wound site prior to using the chimeric skin implant. Depending on the situation, it may be desirable for the chimeric implant to be cultured on the dermal replacement for several days prior to implantation at the wound site. The use of dermal replacement provides a suitable environment for keratinocytes to attach and proliferate. In the case of epithelial substitutes, it is important to derive the dermal foundation for transplantation into severe wounds. That is, it would be optimal if the dermal substrate was readily obtained. Thus, one embodiment of the present invention uses cultured dermal substitutes, such as DermaGraft (Advanced Tissue Science, La Jolla, Calif.) For attachment and proliferation of keratinocytes. For example, dermagraft is a bifactorial neoplastic fibroblast cultured in biodegradable polyglactin and is a permanent dermal replacement. Since the immune response rarely occurs in these fibroblasts, dermagraft rarely produces an immune response.

Three-dimensional chimeric skin implants can be provided in deep wounds (this method of manufacture is only one example and is not limited to this).

(a) placing bifactorial epithelial cells on a biodegradable, biocompatible substrate to grow to about 25-90% confluence;

(b) frozen storage of the bifactorial cells and substrate until use;

(c) obtaining three-dimensional dermal equivalents and placing them on the wound site;

(d) placing the bifactorial cells on a biocompatible substrate and thawing as described above;

(e) obtaining self-recognized epithelial cells and seeding over bifactorial cells;

(f) placing the implant on the dermal equivalent to attach a chimeric skin implant to the wound site;

(g) The chimeric skin implant is covered with a dressing that can be appropriately fixed to prevent mechanical forces or infection from the outside.

According to one embodiment, a chimeric skin substitute is used comprising the following three components. That is, (1) an internal component consisting of a biocompatible dermal structure having a biodegradable or removable backbone as the base material; (2) intermediate components, including self-recognized epithelial cells (eg, keratinocytes or mechanically ground skin) obtained from a patient or self-recognized provider; (3) bifactorial epithelial cells (e.g., keratin forming cells) cultured on dermal constructs comprising biocompatible dermal parts having a biodegradable or removable backbone as a base material with a transmissive sheath such as a BIOBRANE membrane External components, including.

According to a preferred embodiment, the bifactorial cells grow on the dermal part of the outer component and attach to the dermal part (opposite the membrane sheath) and the dermal side of the outer component is inscribed toward the self-recognizing cell side of the intermediate component. .

The complex skin substitute may be used in the treatment of wounds such as burns after being made in vitro. Alternatively, the complex may be made at the same time as the transplant. That is, after placing the internal dermal construct component on the wound site and placing self-recognizing cells on top of the dermal component (immediately or immediately after vascularization), the dermis is directed inward and is two-factor The external component containing the cells is placed on top of the self-recognizing cells. Thus, in order from the outside of the complex skin substitute to the innermost layer, the outermost layer membrane of the outer component, the dermal portion of the outer component containing the bifactorial cells, the self-recognizing inner layer, the innermost dermal replacement layer To achieve.

According to another embodiment, the intermediate component may be composed of cells obtained from a bifactorial source or from a combination of a self-recognizing source and a bifactorial source. In addition, the cells of the intermediate component may be unicellular suspensions, microdermal pieces, ground or dispersed skin or skin in the form of sheets. According to another embodiment, the cells cultured on the external component can be self-recognized cells or a combination of self-recognized cells and divalent cells, wherein a self-recognized protein, a dimeric protein or a combination thereof is present. It can be cultured in an environment that does not exist or in the absence of.

According to a preferred embodiment, the external component is a variant of Dermagraft-TC (aka TRANSCYTE). Dermagraft-TC is a dermal replacement construct that includes dead dermal components attached to a sialastic membrane, such as biobrain (disclosed in US Pat. No. 5,460,939). According to the invention, this variant called transsite comprises a cellular additive or a protein additive. More specifically, the dermal replacement constructs comprise dimeric epithelial cells growing on the constructs. And, it may or may not additionally contain protein components that help the growth and proliferation of the cells.

The external component plays several roles. In other words, it serves as a barrier to control water loss and infection defenses, and keeps other components in place to best attach to the wound in an environment where cells can grow optimally. In addition, the external components are transparent and therefore visible to the naked eye during the course of treatment.

Both internal and external components can be grown or formed in vitro and frozen and stored before being applied to a living body. According to a preferred embodiment, a dead dermagraft is obtained from a patient, mechanically dispersed or crushed, on a self-recognized skin, covered with transsite (preferably on the side facing the wound), incubated with bimodal keratinocytes and modified. Cover or coat with proteinaceous solution. When the patient's skin cells grow until they fuse to the top of the inner dermagraph, the external components are finally removed or sloughed off.

The invention can be described in more detail as follows. However, the following description is for the purpose of describing the present invention, but is not intended to limit the invention. Similar to the following methods and techniques are also applicable to the present invention.

Substrates used for in vitro culture of bifactorial cells

Substrates must be biocompatible as well as capable of degradation in vivo so that they degrade over time and do not need to be removed later. In addition, the substrate, such as a membrane, should be reversibly hydrated to adsorb and / or release the exudate to prevent the exudate from accumulating at the wound site. It is desirable for the membrane to have a small pupil size to reduce or prevent bacterial infection on the wound site. The substrate should be able to be attached to the wound or treated with a bioadhesive so that it can adhere to the wound without damaging epithelial cells or changing the water vapor permeability of the membrane. The substrate should be moderately flexible to accommodate various types of wound surfaces.

According to one embodiment, the bifactorial epithelial cells can be propagated on a hydrophilic synthetic polyurethane membrane dressing, such as Spirofilm, a hydroderm from Wilshire Medical, Dallas or Polymedia, Denver. These two films are very large and varying in water vapor permeability, and part of their surface is covered with a biocompatible adhesive coating. Wetness of the polyurethane membrane increases the water vapor permeability significantly. The water vapor permeability of hydroderm is a function of membrane wetness. Skin typically has a water vapor transmission rate of 200 to 2000 gm ⁻² −d ⁻¹ . In the case of damaged or burned skin, the water vapor transmission ranges from 3000 to 5000 gm ⁻² −d ⁻¹ . Water vapor permeability is between 1500 and 2000 gm ⁻² −d ⁻¹ for conventional hydrophobic polyurethane films with an equilibrium moisture content of less than 1%. However, when coated with a conventional medical adhesive, the adhesive prevents water vapor permeation so that the water vapor permeability drops to 500 to 100 gm ⁻² −d ⁻¹ . The water vapor permeability should not be such that the tissue can be immersed in water, channeling, leaking, and infection.

Another possible hydrophilic membrane is the outer film of Mithraplex, a product of Golden Media, Inc., of Goldden. It is a flexible, transparent polyurethane membrane that permeates water vapor but does not permeate other liquids or microorganisms (Lead, J. Biomet. Appl. 6: 26-31 (1990)).

Cultured bifactorial epithelial cells may be added upside down to the wound site, for example, without the need to enzymatically detach the cells from the membrane. Keratin-forming cells cultured on these membranes have been shown to express a lot of integrins involved in the wound healing process.

Hilaruronic acid membranes can also be used to culture keratinocytes. Hilaruronic acid is known to be biocompatible and its antigenicity is low (Cortibo et al. Biomaterials 12: 727 (1991)). Partial or wholly esterification of hilaluronic acid with an ethyl or benzyl group results in a material having high biocompatibility and a very long or medium half life. For example, LaserSkin, a product of Fidia Advanced Biocopolymer, Abano Terme, Italy, can serve as a carrier system of the cultured bifactorial keratin forming cells of the present invention. This 200 micrometer thick membrane has micropores 40 to 60 micrometers in diameter through which not only steam can pass, but also cells can migrate.

According to another embodiment of the present invention, fibronectin mat is used. Ezim et al. Have reported on the use of fibronectin mat as an in vitro support of keratinocytes (see Biomaterials 14: 742 (1993)). Keratin-forming cells cultured on fibrononectin mat increase the expression of integrins such as integrins α5β1 and αvβ5 in treating wounds (Plazapati et al., 5th Annual Meeting of the European Tissue Repair Society, Padua 1995, Italy 8/9 See month).

Another substrate for the culturing of bifactorial cells is a dermal construct comprising a biocompatible skin portion having a biodegradable or removable backbone as the base material along with a transitional cover such as biobrain. Bifactorial cells may be added to either side of the dermal construct. However, it is desirable to add to the skin side. Bifactorial cells may be cultured with or without additional protein factors in the skin construct.

Another substrate for culturing bifactorial epithelial cells of the invention is fibrin glue or collagen gel. In the present invention, any film or substrate capable of supporting the growth of cultured epithelial cells can be used. Preferably, cells cultured on the membrane or substrate do not have to be enzymatically removed.

When culturing cells in a substrate, such as a membrane, the membrane must remain taut without wrinkles or shrinkage as the cells grow on the membrane.

Another substrate is a hydrogel. It is preferably a polymer composition made from a hydrophilic polymer, in which a natural or synthetic polymer is crosslinked via photopolymerization to form a three-dimensional lattice structure to attract water molecules to form a gel. Polymers useful for forming hydrogels include polysaccharides or carbohydrates such as arginate, polyphosphazine, polyethylene glycol or polyethylene oxide. In the present invention, it is preferable that the hydrogel is polymerized before being added to the dimorphic cells. Further details regarding the preparation of the hydrogel are described in US patent application Ser. No. 08 / 862,740, filed May 25, 1997, which is incorporated herein by reference.

See Renecamp et al., J. Surgical Research 62: 288-295 (1996) for a general overview of the various substrates suitable for culturing keratinocytes.

Formation of Dimorphic Cells on the Matrix

Two-factor epithelial cells were viewed by suitable methods known in the art (see Doyle, A. Griffith, J. B. and Newell, D., "Cell & Tissue Culture: Laboratory Procedures", Willie Press (1995)). It can be cultured on the membrane and the substrate of the invention.

For example, keratinocytes and melanocytes can be separated as follows. (However, not limited to the following method) Tissue samples (e.g., foreskin) are well arranged so that the entire surface is easily exposed to the antibody. For example, subcutaneous tissue was cut out with sterile scissors and about 10 milliliters of PBS containing calcium ions and magnesium ions were added and cell debris was washed. The tissues were washed with concentrated antibody solution and left for 5 minutes. The tissue was transferred to 0.25% trypsin / EDTA and cut longitudinally using surgical scalpels or scissors to make long or square pieces about 3 millimeters wide. The cut tissue contained in the trypsin was placed in a carbon dioxide incubator at 37 ° C., 5% carbon dioxide, 90% humidity for 2.5 to 3 hours. Tissue pieces were removed from the trypsin solution and the cuticle was separated from the dermis using curved tweezers. The epidermis can be placed in a conical tube containing calcium-magnesium-free PBS (about 0.15% trypsin) and used to break down tissues into single cell suspensions. To facilitate this process, the samples were repeatedly asperated with Paster pipat. When the sample appeared to be a single cell suspension, it was centrifuged at 300 × g for about 10 minutes and the cell pellet thus obtained was resuspended by tapping the bottom of the centrifuge tube lightly. About 1 milliliter of keratin forming cell complete culture medium (KCCM) was added to the cells and the tube was lightly beaten to resuspend the pellet. Five milliliters of KCCM was added back here and pipetted up and down several times until the cell suspension became homogeneous. The cells were then transferred to an incubator (eg T150 flask) and the flask was rotated to distribute the cells evenly. The flask was placed in a carbon dioxide incubator at 37 ° C., 5% carbon dioxide, 90% humidity to allow for mitosis of cells.

These bifactorial epithelial cells were added to substrates (eg membranes) in Petri dishes and grown at a temperature of 37 ° C., 5% carbon dioxide, and 90% humidity. Once the appropriate fusion step has been reached, preferably about 25-90%, more preferably about 70-80%, the cells were either stored frozen or used directly for transplantation. Frozen storage of cultured epithelial sheets was performed by methods known in the art (see US Pat. No. 5,145,770 to Tubo et al.). Then, at appropriate time points (generally just before transplantation), thawed dimorphic cells on the stroma were thawed.

According to another embodiment, the bifactorial cells can be genetically engineered prior to culturing to knock out expression of factors that promote an immune response against the transplanted bifactorial cells. Negative modification techniques for lowering the expression level of the gene or the activity of the gene of interest will be described later. As used herein, the term "negative modification" refers to a modification in which the amount and / or activity of the target gene product is further reduced when the modification is performed than when there is no modification. There are several ways to reduce gene expression of bifactorial cells. For example, a standard similar recombination technique can be used to completely inactivate a gene (commonly referred to as "knockout"). In general, exons (or 5 ′ exons of that region) encoding an important portion of a protein are stopped by positive selection labels (eg neo) to inactivate the gene by preventing the production of mRNA from the gene of interest. Part of the gene can also be deleted to inactivate it. Constructs with two regions homologous to the target gene distant in the genome can also be used to delete sequences published between these two regions (Mömbertz et al., 1991, Proc. Acad. Sci. USA 88: 3084-3087).

Antisense and ribozyme molecules that inhibit the expression of the gene of interest may be used to reduce the activity of the gene of interest. For example, antisense RNA molecules that inhibit the expression of histocompatibility gene complex (HLA) have been shown to be most useful in relation to immune responses. Suitable ribozyme molecules can also be found in Hasseloff et al., 1988, Nature 334: 585-591; Sauug et al., 1984, Science 224: 574-578; It may also be prepared according to the method described in Sauug and Sesch, 1986, Science 231: 470-475. Triple-helix molecules can also be used to lower the activity of the gene of interest. More details on these methods can be found in L.G. See Davis et al. Basic Methods in Molecular Biology, 2nd Edition, Apelton & Lange (1994).

One of the techniques described above can be used to modulate the expression of MHC II group molecules in the bifactorial cells of the present invention to lower the risk of rejection of chimeric implants.

For example, after knocking out an undesirable MHC antigen, one or more genes encoding one or more receptor compatible HLA antigens may be transduced and then transplanted into a two-factor cell to enhance or enhance histocompatibility of the transplanted two-factor cell. Can be.

Obtaining Self-Recognized Epithelial Cells

Self-recognized epithelial cells were isolated by the following method (preferably separation is performed immediately before transplantation). Healthy tissue from the self-recognition provider was washed with 70% ethanol, cold probiodine, again 70% ethanol. The washed tissue was chopped to cut the epidermal layer from the dermal layer. The epidermal layer was then washed with cold phosphate buffered saline (PBS) and suspended in 0.25% trypsin-EDTA solution containing 2.5 mg / ml of dispase and mechanically ground for 10 minutes at room temperature. Epithelial pellets were resuspended in appropriate concentrations of PBS. Self-recognition cells were added to cultured bifactorial cells on the surface of the substrate as described above. Or implanted directly into the wound. Thereafter, the cells were covered with a bifactorial cell-substrate construct so as to face the wound as described above.

Transplantation of Living Chimeric Skin Implants

According to one embodiment of the present invention, a cultured skin substitute, such as dermagraft, can be placed on the wound site. Dermagraf can be thawed after freezing storage before use. In this case, wash with sodium chloride solution (0.9% saline). The wound is preferably cleaned by rubbing or washing with sterile solution (surgical iodine such as 0.75% titrateable iodine or 0.9% sodium chloride solution) prior to implantation. After dermagraft on the wound (optionally sutured to the wound), two-factor cells cultured on a biocompatible substrate as described above are inoculated with a self-recognized epithelial cell obtained from a self-recognition provider as described above. do. Self-recognizing cells attach to the surface of the bifactorial cell-substrate construct (ie, cells attach to the surface of the construct containing the bifactorial cells) and invert the construct onto the wound site (i.e., the cells contact the wound and dermagraft). Facing). The wound is then wrapped or covered with a protective dressing and then sutured with sutures.

Claims (50)

A live chimeric skin substitute prepared by a method comprising obtaining a self-recognized epithelial cell from a patient and inoculating the epithelial cell into a biocompatible substrate containing a bifactorial epithelial cell cultured in vitro. The skin substitute of claim 1, wherein the dimeric cells comprise keratinocytes and / or melanin forming cells. The skin substitute of claim 1, wherein the dimeric cells are confluent. The skin substitute of claim 1, wherein the dimeric cells have a confluence of about 25 to 90%. The skin substitute of claim 1, wherein the dimeric cells are genetically engineered cells. The skin substitute of claim 1, wherein the self-recognizing cells comprise keratinocytes and / or melanocytes. The skin substitute of claim 1, wherein the substrate is a biodegradable substrate. 8. The skin substitute of claim 7, wherein the substrate is a hydrophilic synthetic polyurepan membrane, hyaluronic acid membrane, fibronectin mat, fibrin glue, collagen gel or hydrogel. The skin substitute of claim 1, wherein the self-recognizing cells are inoculated at a concentration of about 10 ⁴ / cm ² . The skin substitute of claim 1, wherein the ratio of self-recognized cells to dimeric cells is from 1: 5 to 1:50. The skin substitute of claim 1, wherein said biocompatible substrate containing in vitro cultured dipotent cells is frozen and thawed prior to inoculation with self-recognized cells. A live chimeric skin substitute comprising (a) obtaining self-recognized epithelial cells from a patient, and (b) inoculating the obtained self-recognized cells on a biocompatible substrate containing bigenic epithelial cells cultured in vitro. Manufacturing method. (a) obtaining self-recognized epithelial cells from the patient, (b) inoculating the obtained self-recognized cells on a biocompatible substrate containing bifactorial epithelial cells cultured in vitro to make a chimeric skin substitute, ( c) A method of implanting a live chimeric skin substitute into a wound site comprising inverting the skin substitute and implanting the wound site so that cells face the wound site. (a) obtaining self-recognized epithelial cells from the patient, (b) inoculating the obtained self-recognized epithelial cells at the wound site, and (c) having a biocompatible substrate (in vitro) so that the bifactorial cells face inward toward the self-recognized cells A live chimeric skin substitute in the wound site, comprising inverting and transplanting the wound into a wound site. The method according to any one of claims 12 to 14, wherein the dimeric cells comprise keratinocytes and / or melanin forming cells. The method according to any one of claims 12 to 14, wherein the dimeric cells are confluent. 15. The method of any one of claims 12-14, wherein said dimeric cells have a confluence of about 25-90%. 15. The method of any one of claims 12-14, wherein said dimeric cells are genetically engineered cells. The method of claim 12, wherein the self-recognizing cells comprise keratinocytes and / or melanin forming cells. The method of claim 12, wherein the substrate is a biodegradable substrate. 21. The method of claim 20, wherein the substrate is a hydrophilic synthetic polyurepan membrane, hyaluronic acid membrane, fibronectin mat, fibrin glue, collagen gel or hydrogel. The method of claim 12, wherein the self-recognizing cells are inoculated at a concentration of about 10 ⁴ / cm ² . 15. The method of any one of claims 12-14, wherein the ratio of self-recognized cells to dimeric cells is from 1: 5 to 1:50. The method according to any one of claims 12 to 14, wherein the biocompatible substrates containing in vitro cultured dipotent cells are frozen and thawed before inoculation into the self-recognizing cells. The method of claim 13, wherein the inoculation of the obtained self-recognized cells is carried out at about the same time as when transplanted onto a biocompatible substrate containing cultured bifactorial epithelial cells. The method of claim 13, wherein the inoculated self-recognized cells are transplanted after in vitro culture on a biocompatible substrate containing dimeric epithelial cells. The method of claim 13 or 14, wherein the wound site is a deep wound or a very thick wound. The method of claim 27, further comprising implanting a dermal replacement in the wound site prior to implanting the chimeric skin replacement, wherein the chimeric replacement is inserted such that the cells of the chimeric replacement face inward towards the dermal replacement. (a) an internal component comprising a biocompatible dermal structure having a biodegradable or removable backbone as a base material; (b) an intermediate component comprising epithelial cells; and (c) an external component comprising epithelial cells cultured in vitro on a dermal construct comprising a dermal appendage having a biodegradable or removable backbone as the base material (the dermal portion). Complex skin substitutes, which are associated with the transitional cover and face inward towards the intermediate components of the epithelial cells. 30. The complex skin substitute of claim 29, wherein the dermal construct of the internal component comprises mesenchymal stem cells. The complex skin substitute of claim 29, wherein the epithelial cells of the external component are in vitro cultured on the dermal portion of the construct. 30. The complex skin substitute of claim 29, wherein the transitional covering of the external component is a membrane. 33. The complex skin substitute of claim 32, wherein the membrane is a sialic membrane. 30. The complex skin substitute of claim 29, wherein the epithelial cells of the external component are self-recognizing cells, dimeric cells or combinations thereof. 35. The complex skin substitute of claim 34, wherein the external component is further modified by the addition of a self-recognizing protein, a dimeric protein or a combination thereof. 30. The complex skin substitute of claim 29, wherein the epithelial cells of the intermediate component are in the form of sheets, single cell suspensions, fine skin pieces, or ground or dispersed skin. 30. The complex skin substitute of claim 29, wherein the epithelial cells of the intermediate component are self-recognizing cells, dimeric cells or combinations thereof. 38. The complex skin substitute according to any one of claims 29, 34 and 37, wherein said epithelial cells are keratinocytes and / or melanin forming cells. 38. The complex skin substitute according to any one of claims 29, 34 and 37, wherein said epithelial cells are genetically engineered. (a) an internal component comprising a biocompatible dermal structure having a biodegradable or removable backbone as a base material; (b) an intermediate component comprising epithelial cells; And (c) an external component comprising epithelial cells cultured in vitro on a dermal construct comprising a dermal appendage having a biodegradable or removable backbone as a base material with a transitional sheath, wherein the dermal portion comprises an intermediate component of the epithelial cell. Face-to-face), wherein the complex skin substitute is implanted into the wound site such that the internal component (a) is in direct contact with the core of the wound and the external component (c) is adjacent to the outermost portion of the wound. (a) implanting a biocompatible first internal dermal construct having a biodegradable or removable backbone as a base material at the wound site; (b) obtaining self-recognizing epithelial cells from the patient; (c) inoculating the obtained self-recognizing epithelial cells on top of the internal dermal structures in the wound site; (d) a second outer dermal construct having epithelial cells cultured in vitro and comprising a dermal appendage with a transitional covering and a biodegradable or removable skeleton as a base material, wherein the epithelial cells of the outer dermal construct face the wound site A method for in vivo preparation of a complex skin substitute at a wound site comprising implanting on top of the self-recognizing cells. (a) inoculating epithelial cells on a biocompatible first dermal construct having a biodegradable or removable backbone as the base material, (b) having the cultured epithelial cells and biodegradable or removed as the base material with transitional covering A method of in vitro preparation of a complex skin substitute comprising placing a second dermal construct comprising a dermal appendage with a possible backbone over the first dermal construct such that cells of the second dermal construct face cells of the first dermal construct. 42. The method of claim 41, wherein the epithelial cells of the outer dermal construct are self-recognizing cells, dimeric cells or combinations thereof. 43. The method of claim 42, wherein the epithelial cells of the first dermal construct are self-recognizing cells, dimeric cells or combinations thereof. The method of claim 42, wherein the epithelial cells of the second dermal construct are self-recognizing cells, dimeric cells, or a combination thereof. 43. The method of any one of claims 40-42, wherein said epithelial cells are keratinocytes and / or melanin forming cells. 43. The method of any one of claims 40-42, wherein said epithelial cells are genetically engineered. 43. The method of claim 41 or 42, wherein the epithelial cells of the second dermal construct are cultured in vitro on the dermal section. 43. The method of any one of claims 40-42, wherein the transitional cover is made of a membrane. The method of claim 49, wherein the membrane is a sialic membrane.