Skin substitutes with human dermal blood and lymphatic capillaries in a three-dimensional in vitro system

Abstract

The present invention relates to the production of skin substitutes with a defined ratio of blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) and their use in medicine.

Description

[Technical Field]

[0001] This application claims priority to European Patent Application (EP) No. 22205605.3, filed November 4, 2022, which is incorporated herein by reference.

[0002] The present invention relates to the production of skin substitutes with a defined ratio of blood endothelial cells (BECs) to lymphatic endothelial cells (LECs), and their use in medicine. [Background technology]

[0003] Cells and tissues are essentially dependent on the vasculature to provide vital nutrients and oxygen for metabolic exchange. Thus, with regard to skin, impaired angiogenesis in wounds impedes healing, resulting in, for example, scar-like dermal structures and tissue contraction. Several approaches have been employed to enhance the rate of angiogenesis, including the delivery of proangiogenic growth factors and cell-based therapies.

[0004] The latter strategy, the so-called prevascularization approach, offers an innovative and effective treatment option for promoting wound healing. To this end, microvascular networks with dimensions and characteristics similar to those of the human dermal microvasculature have been incorporated into biocompatible biomaterials, such as collagen type I, fibrin, PEG, and silk scaffolds. These biomaterials have been seeded with human endothelial cells (ECs). To create a prevascularized dermal substitute, the endothelial cells are co-seeded with dermal fibroblasts. Epidermal keratinocytes are then seeded on top of the prevascularized dermal substitute, creating an artificially prevascularized skin substitute. After implantation, the capillaries embedded in the prevascularized skin substitute rapidly connect (anastomose) with the host's vascular and lymphatic systems, respectively.

[0005] This connection process, called coaptation or anastomosis, is characterized by intimate interaction between preformed capillaries and the host vasculature of the wound bed. This can occur as either internal or external coaptation. Internal coaptation occurs within the implant and involves the retraction of the tissue-engineered graft's capillaries, which are then replaced by the invasion of host microvasculature. In contrast, external coaptation is characterized by the extension of the graft's capillaries and their reconnection to the wound bed's microvasculature. Importantly, coaptation allows for reperfusion of the graft within four days, an advantage compared to tissue-engineered grafts without induced angiogenesis, a slow and inefficient process that initially relies entirely on diffusion, resulting in inadequate delivery of oxygen, nutrients, and growth factors. Thus, induced angiogenesis has been shown to promote cell survival, differentiation, and physiological integration of engineered tissues.

[0006] Marino et al. ((2014) Science Translational Medicine, vol. 6, no. 221) disclose a method for isolating HDMECs and fibroblasts from foreskin by scratching and co-culturing them to obtain human skin grafts. Bourland et al. ((2018) Scientific Reports, vol. 8, no. 1, p. 13191) disclose the production of a three-dimensional melanoma model system. HDMECs and fibroblasts are co-isolated by scratching from excised skin of a healthy donor. European Patent (EP) No. 3 174 563 B1 discloses the production of skin grafts from foreskin or adipose tissue samples. HDMECs and fibroblasts are co-isolated and cultured on gelatin-coated dishes. Fibroblasts are removed by scratching. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] European Patent (EP) No. 3 174 563 B1 [Non-patent literature]

[0008] [Non-Patent Document 1] Marino et al.,(2014)Science Translational Medicine,vol.6,no.221 [Non-patent document 2] Bourland et al.,(2018)Scientific Reports,vol.8,no.1,p.13191 Summary of the Invention [Problem to be solved by the invention]

[0009] Based on the above-mentioned state of the art, the object of the present invention is to provide means and methods for treating certain skin diseases and skin defects using skin substitutes. This object is achieved by the subject matter of the independent claims herein, with further advantageous embodiments described in the dependent claims herein, the examples, figures and general description. [Means for solving the problem]

[0010] Summary of the Invention For the isolation of primary cells from a single skin biopsy, we established a method to simultaneously isolate five different cell populations: keratinocytes and melanocytes from the epidermis (top of the skin), and fibroblasts, pericytes, and endothelial cells (ECs) from the dermis (bottom). Most importantly, we were able to separate vascular and lymphatic endothelial cells.

[0011] Fibroblasts and ECs are isolated from the dermis by collagenase type I digestion, followed by enrichment of ECs by magnetic sorting. Subsequently, BEC and LEC fractions of ECs are separated by flow cytometry or magnetic sorting after a short in vitro culture period. BECs represent the CD31-positive, podoplanin-negative fraction of total ECs, while LECs represent CD31-positive, podoplanin-positive cells. Furthermore, BECs can be separated into arterial (neuropilin-1-positive, ephrin-B-positive) and venous (neuropilin-2-positive, ACKR1 (DARC)-positive) cells.

[0012] At the same time, magnetic sorting of CD146 marker can enrich pericytes from the dermal cell fraction.Pericytes, or perivascular / mural cells, lining the dermal microvascular capillaries, play an important role in maintaining vascular stability and vascular remodeling.

[0013] We further describe the use of isolated and cultured cells to prepare angiogenic skin substitutes with varying ratios of BECs to LECs, which may also contain pericytes, fibroblasts, melanocytes, and keratinocytes, resulting in angiogenic skin substitutes intended to support the healing of cutaneous wounds in various skin diseases.

[0014] In particular, skin substitutes containing 100% / 0% BEC / LEC may be applied to indications such as ischemic (arterial) ulcers, venous leg ulcers, or acute burns.

[0015] Skin substitutes containing 0% / 100% BEC / LEC can be applied to indications such as secondary lymphedema, lipedema, elephantiasis (lymphatic filariasis), or lymphangioleiomyomatosis.

[0016] Skin substitutes containing a mixed ratio of BEC / LEC (50% / 50%) can be applied, for example, in lymphedema and skin reconstruction surgery.

[0017] A first aspect of the present invention relates to a method for producing a skin substitute, said method comprising the steps of: a. providing a skin tissue sample isolated from a mammalian subject; b. separating the skin tissue sample into the dermis and epidermis; c. enzymatically degrading the matrix component of the dermis to obtain a plurality of cells in suspension; d. In a CD31 step, the plurality of cells are subjected to CD31 + Cell fractions and CD31 - sorting into cell fractions; e. CD31 + The cell fraction was cultured in an appropriate medium under appropriate cell culture conditions to express multiple CD31 + Obtaining cells; f. In the BEC / LEC step, the plurality of CD31 +sorting the cells into vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs); g. In a culturing step, the BECs and / or LECs are cultured in an appropriate medium under appropriate cell culture conditions to obtain a skin substitute.

[0018] A second aspect of the invention relates to a skin substitute obtainable by the method according to the first aspect.

[0019] A further aspect of the present invention relates to a skin substitute according to the second aspect for use in the treatment of certain skin disorders.

[0020] Terms and Definitions For the purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa. In the event that a definition set forth below conflicts with any document incorporated herein by reference, the definition set forth herein shall control.

[0021] As used herein, the terms "comprising," "having," "containing," "including," and other similar forms, and their grammatical equivalents, are intended to be equivalent in meaning and to be open-ended in that the listing of one or more items following any one of these words does not imply an exhaustive listing of such one or more items, or that it is limited to only the listed item or items. For example, an item "comprising" components A, B, and C can consist of components A, B, and C (i.e., contain only components A, B, and C), or it can include not only components A, B, and C, but also one or more other ingredients. Thus, "comprising" and its similar forms, and its grammatical equivalents, are intended and understood to include disclosure of "consisting essentially of" or "consisting of" embodiments.

[0022] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where one or both of the limits are included in the stated range, ranges excluding either or both of those included limits are also included in the disclosure.

[0023] As used herein, reference to "about" a value or parameter includes (and describes) a variation on the value or parameter itself. For example, a statement referring to "about X" also includes the statement "X."

[0024] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural references unless the context clearly dictates otherwise.

[0025] As used herein, "and / or" is considered to specifically describe each of the two specified features or components with or without the other features or components. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A alone," and "B alone." Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0026] The term "when grafted" relates to the state of the skin substitutes disclosed herein under post-graft conditions and can alternatively be read as "when grafted."

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard procedures are used for molecular, genetic, and biochemical procedures (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th ed., John Wiley & Sons, Inc.) and chemical procedures.

[0028] Cell biology, diagnostic method inventions: markers, ligands As used herein, the term "positive," when used in the context of marker expression, refers to expression of an antigen as assayed by a fluorescently labeled antibody, and the fluorescence of the label in structures (e.g., cells) that are "positive" is at least 30% higher (≧30%), particularly ≧50% or ≧80%, in median fluorescence intensity compared to staining with an isotype-matched fluorescently labeled antibody that does not specifically bind to the same target. Expression of such markers is indicated by a superscript "plus" ( + ) (e.g., CD31 + ), or by placing a plus sign after the name of the marker ("CD31+"). When the term "expression" is used herein in the context of "gene expression" or "marker or biomolecule expression" and no further qualification of "expression" is mentioned, this includes the meaning of "positive expression" as defined above.

[0029] As used herein, the term "negative," when used in the context of marker expression, refers to expression of an antigen assayed by a fluorescently labeled antibody in which the median fluorescence intensity is less than 30%, particularly less than 15%, higher than the median fluorescence intensity of an isotype-matched antibody that does not specifically bind to the same target. Expression of such markers is indicated by a superscript minus ( - ) (e.g., CD31 - ), or by placing a minus sign after the marker name ("CD31-").

[0030] High expression of a marker, e.g., high expression of NRP1, refers to the amount of expression of a marker that exhibits the highest fluorescence intensity per cell compared to other populations characterized by lower fluorescence intensity per cell in distinct cell populations detected by FACS. High expression, e.g., NRP1 high The term "highly expressed" refers to the same property.

[0031] Low expression of a marker, e.g., low expression of NRP1, refers to the expression level of a marker that exhibits the lowest fluorescence intensity per cell compared to other populations characterized by higher fluorescence intensity per cell in distinct cell populations detected by FACS. low The term "lowly expressed" refers to the same property as "low" or "lo" in superscript after the name of the marker, such as:

[0032] Marker expression can be assayed via techniques such as fluorescence microscopy, flow cytometry, ELISPOT, ELISA, or multiplex analysis.

[0033] The term "skin substitute" herein relates to a three-dimensional scaffold of skin tissue produced in the laboratory according to the present invention. Skin substitutes are ex vivo cultured skin tissues. The term "skin mimic" is equivalent to the term skin substitute. Skin substitutes can be of autologous or allogenic origin.

[0034] The term "skin tissue sample" as used herein refers to a sample that contains, at a minimum, the epidermis and dermis. These two tissues are sufficient to isolate all five cell types described below. The minimum sample size requirement is a 4 mm (punch) or larger biopsy (a 4 mm diameter punch biopsy needle is commonly used in dermatology to remove skin pieces for histopathological examination).

[0035] The term "dermis or dermal portion of a replacement" as used herein refers to the layer of skin between the epidermal tissue and the subcutaneous tissue. The structural components of the dermis are collagen, elastic fibers, and an extrafibrous matrix. Nerves, lymphatic vessels, and blood vessels are present in the dermis.

[0036] As used herein, the term "epidermis or epidermal portion of a replacement" refers to the outermost of the three layers that make up the skin. The epidermis is composed primarily of keratinocytes, but may also contain melanocytes.

[0037] The term "relative amount of BECs and LECs" in this specification relates to a predetermined ratio between BECs and LECs. The determination of the ratio is made depending on the purpose of the skin substitute. Each indication for applying the skin substitute according to the present invention requires a defined ratio of BECs and LECs ranging from 100% BECs and 0% LECs to 0% BECs and 100% LECs.

[0038] The term "FACS" as used herein refers to fluorescence-activated cell sorting.

[0039] Any patent documents cited herein are deemed to be incorporated herein by reference in their entirety.

[0040] As used herein, the term "treating" or "treatment" of any disease or disorder (e.g., a skin disorder) refers, in one embodiment, to ameliorating the disease or disorder (e.g., delaying, preventing, or alleviating the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible to the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Methods for assessing the treatment and / or prevention of disease are generally known in the art, except as specifically described below herein. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description of the Invention A first aspect of the present invention relates to a method for producing a skin substitute, the method comprising the steps of: In step a, a skin tissue sample isolated from a mammalian subject is provided, and the isolated sample is supplemented with an antibiotic and a disinfectant.

[0042] In step b, the skin tissue sample is separated into the dermis and the epidermis. In certain embodiments, the separation is achieved using forceps. In certain embodiments, the separation is achieved using a microscope. In certain embodiments, the separation is achieved without the use of an optical device.

[0043] In step c, the matrix components of the dermis are enzymatically digested to obtain a number of cells in suspension. This step can take approximately 20 minutes to 2 hours (depending on the size of the sample). Digestion is required to destroy the so-called extracellular matrix in which the endothelial cells are located and to disrupt the connections between the cells, obtaining single cells that can then be sorted.

[0044] In step d, also referred to herein as the CD31 step, the plurality of cells are subjected to CD31 + Cell fractions and CD31 - The cells are sorted into cell fractions. This can be achieved by a common cell separation method that distinguishes cells by the expression or non-expression of a cell surface marker (here, CD31).

[0045] In step e, CD31 + The cell fraction was cultured in an appropriate medium under appropriate cell culture conditions to express multiple CD31 + Obtain cells.

[0046] In step f, also referred to as the BEC / LEC step, multiple CD31 + The cells are sorted into vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs).

[0047] In step g, also referred to as the culturing step, a plurality of cells including BECs and / or LECs are cultured in a suitable medium under suitable cell culture conditions to obtain a skin substitute.

[0048] Suitable cell culture conditions comprise a temperature of about 37° C., a CO 2 concentration of about 5%, and a relative humidity of about 95%. In certain embodiments, the cells are cultured in endothelial cell medium.

[0049] In certain embodiments, the relative amounts of BECs and LECs used in the culturing step range from 100% BECs and 0% LECs to 0% BECs and 100% LECs. In certain embodiments, the plurality of cells comprising BECs and / or LECs in the culturing step consists of 100% BECs. In certain embodiments, the plurality of cells comprising BECs and / or LECs in the culturing step consists of 100% LECs. In certain embodiments, the plurality of cells comprising BECs and / or LECs in the culturing step includes BECs and other specified cells described below, but does not include LECs. In certain embodiments, the plurality of cells comprising BECs and / or LECs in the culturing step includes LECs and other specified cells described below, but does not include BECs.

[0050] In certain embodiments, the relative amounts of BECs and LECs used in the culture step are between 70% BECs and 30% LECs to 30% BECs and 70% LECs.

[0051] In certain embodiments, the relative amounts of BECs and LECs are selected depending on the purpose of the skin substitute, meaning that each indication to be treated with the skin substitute of the present invention has its own unique challenges and requires a suitable amount of BECs and / or LECs.

[0052] In certain embodiments, the CD31 process is performed via magnetic sorting.

[0053] In certain embodiments, the CD31 process is performed via paramagnetic particles. In certain embodiments, the paramagnetic particles are uniform polystyrene spherical beads that have been made magnetically susceptible. Paramagnetic particles allow for gentle binding of low-abundance antigens or rare cell populations. This method exerts minimal shear forces on the cells compared to MACS and FACS.

[0054] In certain embodiments, the diameter of the paramagnetic particles is 1-5 μm. Generally, using paramagnetic particles as a first step is the most gentle on cells (cell survival) when isolated from skin tissue.

[0055] In certain embodiments, the BEC / LEC process is performed via fluorescence-based or magnetic sorting, hi certain embodiments, the BEC / LEC process is performed via FACS.

[0056] In certain embodiments, in the BEC / LEC step, the BEC-specific marker is selected from the group consisting of PLVAP and PAL-E.

[0057] In certain embodiments, in the BEC / LEC step, the LEC-specific marker is selected from the group consisting of podoplanin, Lyve1, and VEGFR3.

[0058] In certain embodiments, the skin tissue sample is derived from non-fetal tissue. In certain embodiments, the non-fetal tissue is derived from the same patient as the patient receiving the skin substitute. The advantage of using tissue derived from the same patient as the patient receiving the skin substitute is that the skin substitute will not undergo immune rejection.

[0059] In certain embodiments, the non-fetal tissue is derived from surgical removal of the foreskin. In certain embodiments, the skin tissue sample is derived from fetal tissue. In certain embodiments, the fetal skin tissue is obtained from spina bifida surgery. The advantage of using tissue from a tissue donor is that the skin substitute is ready to use, whereas autologous cultured skin substitutes require approximately three weeks of preparation time. Additionally, in most cases, skin tissue donors are very young, and fetal skin tissue or pediatric tissue is more likely to differentiate into specific cell types, resulting in the skin substitute being better integrated into the host's skin.

[0060] In certain embodiments, the skin tissue sample is derived from human tissue.

[0061] In certain embodiments, the skin tissue sample is derived from porcine tissue. The advantage of using porcine tissue as a skin substitute is that more donor tissue is available, and the risk of transmitting infection from the skin donor to the recipient is reduced. In certain embodiments, the skin donor pig is genetically modified so that its skin is less immunogenic and less likely to cause immune rejection in the recipient.

[0062] In certain embodiments, after the BEC / LEC step, the BECs are sorted into arterial and venous endothelial cells via specific markers of arterial and / or venous endothelial cells.

[0063] In certain embodiments, the arterial endothelial cell-specific markers are ephrin B2 positivity and neuropilin 1 (NRP1) high is selected from the group consisting of:

[0064] In certain embodiments, the venous endothelial cell-specific markers are ACKR1 (DARC) positivity and neuropilin 2 (NRP2) high is selected from the group consisting of:

[0065] In certain embodiments, during the BEC / LEC process, additional CD146 + Parietal cells (pericytes) express CD31 - They are sorted from the cell fraction (by expression of CD146) and added during the culture process.

[0066] In certain embodiments, keratinocytes are isolated from the epidermis and added to the culture step.

[0067] In certain embodiments, the skin sample is first cut into small pieces and digested in about 12 U / ml of dispase overnight at 4° C. The epidermis is then mechanically separated from the dermis using forceps.

[0068] In certain embodiments, keratinocytes are isolated by treating the epidermis with about 0.5% (w / v) trypsin solution, or by sorting epidermal cells into the MC1R-negative / E-cadherin-positive fraction (a fraction containing keratinocytes).

[0069] In certain embodiments, melanocytes are isolated from the epidermis and the melanocytes are added in a culture step.

[0070] In certain embodiments, melanocytes are isolated by treatment with about 0.05% (w / v) trypsin solution. In certain embodiments, melanocytes are isolated by sorting epidermal cells into the MC1R-positive / E-cadherin-positive fraction (a fraction containing melanocytes).

[0071] Furthermore, to separate the melanocytes / keratinocytes, the epidermis is digested to separate the cells from the so-called basement membrane and also from each other.

[0072] In certain embodiments, digestion of the tissue sample is carried out with an enzyme or combination of enzymes selected from the group consisting of collagenase II, and / or collagenase IV, and / or a mixture of collagenases I and II commercially available as "Liberase," and / or trypsin.

[0073] In certain embodiments, the culturing step further comprises: - collagen, and / or - fibrin, and / or - gelatin, and / or - PEG, and / or - Polyurethane In certain embodiments, collagen is further added during the culturing step. In certain embodiments, fibrin is further added during the culturing step. In certain embodiments, gelatin is further added during the culturing step. In certain embodiments, PEG is further added during the culturing step. In certain embodiments, polyurethane is further added during the culturing step.

[0074] In a specific embodiment, to prepare an angiogenic hydrogel, collagen type I (bovine collagen type I) is mixed with a cell suspension of a 1:1 mixture of HDMECs and fibroblasts.

[0075] A second aspect of the present invention relates to a skin substitute obtainable by the method according to the first aspect. In certain embodiments, the skin substitute according to the second aspect comprises endothelial cells. In certain embodiments, the dermal portion of the skin substitute according to the second aspect comprises 35% or more endothelial cells. In certain embodiments, the dermal portion of the skin substitute according to the second aspect comprises about 50% endothelial cells. In certain embodiments, all cells of the skin substitute are derived from a single donor subject.

[0076] In certain embodiments, the skin substitute is an isolated skin substitute, i.e., the skin substitute is cultured in vitro and is distinguishable from a skin sample.

[0077] In certain embodiments, the skin substitute, when transplanted into a recipient subject, increases the CD31 + In certain embodiments, the donor's CD31 + More than 15% of blood capillaries express CD31 in recipient subjects within 4 days of transplantation into the recipient subject. + They are able to connect with capillaries, a feature present in skin substitutes containing BECs.

[0078] In certain embodiments, when the skin substitute is transplanted into a recipient subject, it can induce capillaries of the recipient subject to ingrow throughout the dermis and down to the basement membrane. In certain embodiments, when the skin substitute is transplanted into a recipient subject, it can induce capillaries of the recipient subject to ingrow throughout the dermis and down to the basement membrane one week after transplantation into the recipient subject. This characteristic is present in all skin substitutes.

[0079] In certain embodiments, the skin substitute, when implanted into a recipient subject, is capable of attracting capillary ingrowth in the recipient subject for 4 days after implantation into the recipient subject, a characteristic present in all skin substitutes.

[0080] In certain embodiments, the skin substitute, when transplanted into a recipient subject, can exhibit an oxygen saturation of greater than 50% one week after transplantation into the recipient subject, a characteristic present in skin substitutes comprising BECs.

[0081] In certain embodiments, the skin substitute, when transplanted into a recipient subject, is capable of having an oxygenated hemoglobin content of greater than 30% one week after transplantation into the recipient subject, a characteristic present in skin substitutes comprising BECs.

[0082] In certain embodiments, the skin substitute, when transplanted into a recipient subject, can comprise a greater number of M2 macrophages than M1 macrophages three weeks after transplantation into the recipient subject, a characteristic present in skin substitutes comprising BECs.

[0083] In certain embodiments, the skin substitute, when transplanted into a recipient subject, exhibits low or no expression of CK16 and CK17 (CK16) at 2 weeks after transplantation into the recipient subject, particularly at 1 week after transplantation into the recipient subject. low and CK17 low or CK16- and CK17 - This characteristic is present in skin substitutes containing BECs.

[0084] In certain embodiments, the ratio of cell types in the dermis is selected from Table 1, the ratio of arterial to venous BECs is selected from Table 2, and the ratio of cell types in the epidermis is selected from Table 3. In certain embodiments, the ratio of cell types in the dermis is selected from Table 1, and no epidermal cells are used.

[0085] Differences exist between existing skin substitutes and capillaries in morphology, structure, and function. For example, capillaries have a continuous basement membrane, pericyte coverage, and express specific markers (collagen IV), are specialized for transporting blood, and are involved in the exchange of oxygen and nutrients, whereas lymphatic capillaries do not have a basement membrane, are not pericyte coverage, express other specific markers (Lyeve1, podoplanin), and take up excess fluid. The latter can be measured using the Evans Blue assay.

[0086] Generally, the skin substitute provided by the present invention can be used to treat skin defects caused by burns or in reconstructive skin surgery. The present invention is also useful for treating skin diseases or chronic wounds.

[0087] A further aspect of the invention relates to a skin substitute according to the second aspect for use in the treatment of secondary lymphoedema. In a particular embodiment, the skin substitute for use in the treatment of secondary lymphoedema is cultured from 0% BECs and 100% LECs in a BEC / LEC relative amount.

[0088] Secondary lymphedema is caused by damage to the lymphatic system or problems with the movement and drainage of fluids through the lymphatic system, which can be caused by cancer treatment, infection, injury, limb inflammation, or limb immobility. Secondary lymphedema is caused by damage or blockage of the lymphatic system and most frequently occurs as a result of cancer treatment, such as in breast cancer survivors, but is also a common complication of other solid tumors. In most cases, the onset of postoperative lymphedema occurs several months, sometimes even years, after initial treatment, and occurs in a delayed but persistent manner. Therefore, skin substitutes with artificially engineered lymphatic capillaries can remove excess interstitial fluid from damaged areas (e.g., burns) and reduce cutaneous lymphedema.

[0089] A further aspect of the present invention relates to a skin substitute according to the second aspect for use in the treatment of chronic edema. In a particular embodiment, the skin substitute for use in the treatment of chronic edema is cultured from about 50% BECs and about 50% LECs in a BEC / LEC relative amount.

[0090] Chronic edema in the lower extremities is usually a manifestation of lymphedema, a combination of venous and lymphatic dysfunction.

[0091] A further aspect of the invention relates to a skin substitute according to the second aspect for use in treating acute burns. In a particular embodiment, the skin substitute for use in treating acute burns is LEC-free and has been cultured entirely from BEC cells (the BEC / LEC relative amount is 100% BEC and 0% LEC).

[0092] Burns are caused by skin damage due to excessive heat or other injuries. Heat can be the result of thermal, electrical, chemical, or electromagnetic energy. Ischemic necrosis develops within hours of a burn injury and is most pronounced in tissues directly involved in the burn, but can also develop in distant uninjured tissues, such as muscle, intestine, and lungs. Furthermore, hematological changes impair the microvasculature, thereby impairing blood perfusion in burned tissue. Therefore, skin substitutes with artificially created arterial and venous capillaries can supply oxygen and nutrients to the wound area, thereby improving blood flow and promoting skin healing.

[0093] A further aspect of the present invention relates to a skin substitute according to the second aspect for use in treating arterial ulcers. In a particular embodiment, the skin substitute for use in treating arterial ulcers is cultured from a BEC / LEC relative amount of 100% BECs and 0% LECs. Thus, the skin substitute with artificially generated arterial capillaries can supply oxygen and nutrients to the wound area, thereby improving angiogenesis, healing, and wound closure.

[0094] Arterial ulcers, also known as ischemic ulcers, are caused by poor perfusion / circulation (supply of nutrient-rich blood) within the arteries of the leg. Poor circulation can be caused by diabetes, inflammation, fatty blockages, clogged arteries, peripheral arterial disease (PAD), or infection. When blood does not reach the limb, the area does not receive enough oxygen and nutrients. This causes tissue damage and even cell death. Damaged, blood-starved tissue is unable to heal. Untreated ischemic ulcers can lead to gangrene and limb loss.

[0095] A further aspect of the invention relates to a skin substitute according to the second aspect for use in treating venous leg ulcers, in a particular embodiment the skin substitute for use in treating venous leg ulcers has been cultured from a BEC / LEC relative amount of 100% BECs and 0% LECs.

[0096] Venous leg ulcers are the most common type of leg ulcer, accounting for over 90% of all cases. They typically develop on the inside of the leg just above the ankle and are chronic, long-lasting, open wounds. Ulcers are caused by diseases of the leg veins, such as varicose veins, or as a complication of deep vein thrombosis (DVT). Venous ulcers typically develop when persistent venous hypertension damages the valves in the veins of the leg, resulting in the formation of an ulcer at the ankle. Therefore, skin substitutes with artificially created venous capillaries can improve venous return of deoxygenated blood from the wound area, thereby improving general body blood flow.

[0097] A further aspect of the invention relates to a skin substitute according to the second aspect for use in the treatment of lymphatic filariasis. In a particular embodiment, the skin substitute for use in the treatment of lymphatic filariasis has been cultured from 0% BECs and 100% LECs in a relative amount of BEC / LEC.

[0098] Elephantiasis (lymphatic filariasis) is a painful and disfiguring disease caused by parasitic roundworms that occupy and block lymphatic vessels. It is caused by infection with a parasitic worm that belongs to the nematode family. In areas where filariasis is transmitted, people of all ages are affected. Infection can occur in childhood, but visible symptoms such as swelling of the hands and feet can appear later in life, potentially causing temporary or permanent disability. Therefore, skin substitutes with artificially created lymphatic capillaries could take up excess interstitial fluid in the limbs, thus reducing limb swelling.

[0099] A further aspect of the invention relates to a skin substitute according to the second aspect for use in treating phlebolymphedema. In a particular embodiment, the skin substitute for use in treating phlebolymphedema is cultured from about 50% BECs and about 50% LECs in a BEC / LEC relative amount.

[0100] Venous lymphedema is swelling of mixed etiology due to chronic venous and lymphatic insufficiency. Therefore, when either of these two systems fails to function normally (e.g., chronic venous hypertension, lymphedema), this interdependence leads to a new pathology that simultaneously affects both systems, known as phlebolymphedema, also known as venolymphatic edema. Therefore, skin substitutes with artificially created capillaries / lymphatic capillaries can simultaneously deliver oxygen and nutrients while removing excess interstitial fluid from the limbs, improving blood flow and reducing tissue swelling.

[0101] A further aspect of the invention relates to a skin substitute according to the second aspect for use in the treatment of lymphangioleiomyomatosis. In a particular embodiment, the skin substitute for use in the treatment of lymphangioleiomyomatosis is cultured from 0% BECs and 100% LECs in a BEC / LEC relative amount.

[0102] In people with lymphangioleiomyomatosis, lymphatic vessels can rupture or become blocked (obstructed), causing a buildup of chyle within the chest cavity (chylothorax), a rare but serious condition in which lymphatic fluid (chyle) formed in the digestive system accumulates within the chest cavity.

[0103] A further aspect of the present invention relates to a skin substitute according to the second aspect for use in treating a skin defect, wherein the skin substitute is cultured from a relative amount of BECs / LECs present in the skin of a defect, such as, but not limited to, skin trauma following chemical burns, radiation, or abrasion. A small skin biopsy from a patient is analyzed for specific blood and lymphatic vessel ratios, and the skin substitute is prepared to contain a patient-specific ratio of BECs to LECs, and the skin substitute is applied to the wound.

[0104] [Table 1]

[0105] [Table 2]

[0106] [Table 3]

[0107] In the process of manufacturing a skin substitute, the dermis is first constructed by cell culture of (a) BECs and / or LECs, (b) fibroblasts, and (c) optionally, pericytes. After about one week of dermal culture, the epidermal portion is added to the culture, which consists of keratinocytes and optionally melanocytes. In certain embodiments, the dermis is sufficient to produce a skin substitute, and no epidermal cells are added.

[0108] medical treatment Also included within the scope of the present invention is a method or method for treating a skin condition in a patient in need thereof, comprising administering to the patient a skin substitute in accordance with the above description.

[0109] Manufacturing and treatment methods according to the present invention The present invention further encompasses, as a further aspect, the use of a skin substitute as specified herein for use in a method for the manufacture of a medicament for the treatment or prevention of a skin disorder.

[0110] Similarly, the present invention encompasses a method of treating a patient diagnosed with a skin disorder associated with open wounds, the method comprising administering to the patient a skin substitute as identified herein.

[0111] Where alternative forms of a single separable feature, such as a ratio or a marker protein, or an indication, are described herein as "embodiments," it is understood that such alternative forms can be freely combined to form separate embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a ratio can be combined with any of the alternative embodiments for a marker protein, and these combinations can be combined with any medical indication referred to herein.

[0112] The present invention further encompasses the following:

[0113] item Item 1. A method for producing a skin substitute, the method comprising the steps of: a. providing a skin tissue sample isolated from a mammalian subject; b. separating the skin tissue sample into the dermis and epidermis; c. enzymatically degrading the matrix component of the dermis to obtain a plurality of cells in suspension; d. In a CD31 step, the plurality of cells are subjected to CD31 + Cell fractions and CD31 - sorting into cell fractions; e. CD31 + The cell fraction was cultured and multiple CD31 + Obtaining cells; f. In the BEC / LEC step, the plurality of CD31 + sorting the cells into vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs); g. A step of culturing BECs and / or LECs to obtain a skin substitute in a culturing step. The method comprising:

[0114] Item 2. The method according to Item 1, wherein the relative amounts of BECs and LECs used in the culture step range from 100% BECs and 0% LECs to 0% BECs and 100% LECs.

[0115] Item 3. The method according to Item 1 or 2, wherein the relative amounts of BECs and LECs used in the culture step are between 70% BECs and 30% LECs and 30% BECs and 70% LECs.

[0116] Item 4. The method according to any one of Items 1 to 3, wherein the relative amounts of BECs and LECs are selected depending on the purpose of the skin substitute.

[0117] Item 5. The method according to any one of Items 1 to 4, wherein the CD31 step is carried out via magnetic sorting.

[0118] Item 6. The method according to any one of Items 1 to 5, wherein the CD31 step is carried out via paramagnetic particles.

[0119] Item 7. The method according to any one of Items 1 to 6, wherein the BEC / LEC step is carried out via fluorescence-based sorting or magnetic sorting, particularly FACS.

[0120] Item 8. The method according to any one of Items 1 to 7, wherein in the BEC / LEC step, the BEC-specific marker is selected from the group consisting of PLVAP and PAL-E.

[0121] Item 9. The method according to any one of Items 1 to 8, wherein in the BEC / LEC step, the LEC-specific marker is selected from the group consisting of podoplanin, Lyve1, and VEGFR3.

[0122] Item 10. The method according to any one of Items 1 to 9, wherein the skin tissue sample is derived from non-fetal tissue.

[0123] Item 11. The method according to any one of Items 1 to 9, wherein the skin tissue sample is derived from fetal tissue.

[0124] Item 12. The method according to any one of Items 1 to 11, wherein the skin tissue sample is derived from human tissue.

[0125] Item 13. The method according to any one of Items 1 to 11, wherein the skin tissue sample is derived from porcine tissue.

[0126] Item 14. The method according to any one of Items 1 to 13, wherein after the BEC / LEC step, the BECs are sorted into arterial endothelial cells and venous endothelial cells via an arterial endothelial cell-specific marker and / or a venous endothelial cell-specific marker.

[0127] Item 15. Arterial endothelial cell-specific markers are ephrin B2 positivity and neuropilin 1 (NRP1) high Item 15. The method of item 14, selected from the group consisting of:

[0128] Item 16. Venous endothelial cell-specific markers are ACKR1 (DARC) positive and neuropilin 2 (NRP2) positive. high 16. The method according to item 14 or 15, selected from the group consisting of:

[0129] Item 17. During the BEC / LEC process, CD146 + Parietal cells are CD31 - 17. The method according to any one of items 1 to 16, wherein the cell fraction is sorted from the cell fraction and added in the culture step.

[0130] Item 18. Keratinocytes from the epidermis: by treating the epidermis with a trypsin solution of about 0.5% (w / v); or - by sorting cells of the epidermis into the MC1R-negative / E-cadherin-positive fraction; 18. The method according to any one of items 1 to 17, wherein the isolated keratinocytes are added in a culture step.

[0131] Item 19. Melanocytes originate from the epidermis: by treatment with a trypsin solution of about 0.05% (w / v); or - by sorting epidermal cells into the MC1R-positive / E-cadherin-positive fraction; 19. The method according to any one of items 1 to 18, wherein the isolated melanocytes are added in a culture step.

[0132] Item 20. The method according to any one of Items 1 to 19, wherein the digestion of the tissue sample is carried out using an enzyme or a combination of enzymes selected from the group consisting of collagenase II, and / or collagenase IV, and / or a mixture of collagenase I and collagenase II, and / or trypsin.

[0133] Item 21. In the culturing step, - collagen, and / or - fibrin, and / or - gelatin, and / or - PEG, and / or - Polyurethane 21. The method according to any one of items 1 to 20, wherein

[0134] Item 22. A skin substitute obtainable by the method according to any one of items 1 to 21, wherein the skin substitute particularly comprises endothelial cells, more particularly the dermal portion of the skin substitute comprises 35% or more endothelial cells.

[0135] Item 23. The skin substitute according to Item 22 for use in the treatment of secondary lymphedema.

[0136] Item 24. A skin substitute for use according to Item 23, wherein the skin substitute is cultured from 0% BECs and 100% LECs in a BEC / LEC relative amount.

[0137] Item 25. The skin substitute according to Item 22 for use in treating chronic edema.

[0138] Item 26. A skin substitute for use according to Item 25, wherein the skin substitute is cultured from about 50% BECs and about 50% LECs in a BEC / LEC relative amount.

[0139] Item 27. The skin substitute according to Item 22 for use in treating acute burns.

[0140] Item 28. A skin substitute for use according to Item 27, wherein the skin substitute is cultured from 100% BECs and 0% LECs in relative amounts of BEC / LECs.

[0141] Item 29. The skin substitute according to Item 22 for use in the treatment of arterial ulcers.

[0142] Item 30. A skin substitute for use according to Item 29, wherein the skin substitute is cultured from 100% BECs and 0% LECs in relative amounts of BEC / LECs.

[0143] Item 31. The skin substitute according to Item 22 for use in treating venous leg ulcers.

[0144] Item 32. A skin substitute for use according to Item 31, wherein the skin substitute is cultured from 100% BECs and 0% LECs in relative amounts of BEC / LECs.

[0145] Item 33. The skin substitute according to Item 22 for use in the treatment of lymphatic filariasis.

[0146] Item 34. The skin substitute for use according to Item 33, wherein the skin substitute is cultured from 0% BECs and 100% LECs in a BEC / LEC relative amount.

[0147] Item 35. The skin substitute according to Item 22 for use in the treatment of venous lymphedema.

[0148] Item 36. A skin substitute for use according to Item 35, wherein the skin substitute is cultured from about 50% BECs and about 50% LECs in a BEC / LEC relative amount.

[0149] Item 37. The skin substitute according to Item 22 for use in the treatment of lymphangioleiomyomatosis.

[0150] Item 38. A skin substitute for use according to Item 37, wherein the skin substitute is cultured from 0% BECs and 100% LECs in a BEC / LEC relative amount.

[0151] Item 39. The skin substitute according to Item 22 for use as a pharmaceutical.

[0152] Item 40. When a skin substitute is transplanted into a recipient subject, the recipient subject's CD31 expression level is increased within 4 days after transplantation. + It is possible to connect with capillaries, especially CD31 of the skin substitute donor. + More than 15% of capillaries were found to be CD31 positive in recipient subjects within 4 days of transplantation. + 23. The skin substitute according to item 22, which is connectable to blood capillaries.

[0153] Item 41. The skin substitute according to Item 22, wherein the skin substitute, when transplanted into a recipient subject, is capable of inducing the ingrowth of capillaries of the recipient subject throughout the dermis and down to the basement membrane, particularly, the skin substitute, when transplanted into a recipient subject, is capable of inducing the ingrowth of capillaries of the recipient subject throughout the dermis and down to the basement membrane one week after transplantation into the recipient subject.

[0154] Item 42. The skin substitute according to Item 22, wherein the skin substitute, when transplanted into a recipient subject, is capable of inducing capillary ingrowth in the recipient subject for 4 days after transplantation into the recipient subject.

[0155] Item 43. The skin substitute according to Item 22, wherein the skin substitute, when transplanted into a recipient subject, is capable of exhibiting an oxygen saturation of greater than 50% one week after transplantation into the recipient subject.

[0156] Item 44. The skin substitute according to Item 22, wherein the skin substitute, when transplanted into a recipient subject, is capable of having an oxygenated hemoglobin level that is greater than 30% one week after transplantation into the recipient subject.

[0157] Item 45. The skin substitute according to Item 22, wherein the skin substitute, when transplanted into a recipient subject, is capable of containing a greater number of M2 macrophages than the number of M1 macrophages 3 weeks after transplantation into the recipient subject.

[0158] Item 46. When a skin substitute is transplanted into a recipient subject, it should show low or no expression of CK16 and CK17 (CK16) two weeks after transplantation into the recipient subject, and especially one week after transplantation into the recipient subject. low and CK17 low or CK16 - and CK17 - 23. The skin substitute according to item 22, characterized in that

[0159] Item 47. The skin substitute according to Item 22, wherein the ratio of cell types in the dermis is selected from Table 1, the BEC ratio between arteries and veins is selected from Table 2, and the ratio of cell types in the epidermis is selected from Table 3.

[0160] Item 48. When a skin substitute is transplanted into a recipient subject, the recipient subject's CD31 expression level is increased within 4 days after transplantation. + It is possible to connect with capillaries, especially CD31 of the skin substitute donor. + More than 15% of capillaries were found to be CD31 positive in recipient subjects within 4 days of transplantation. + 23. The skin substitute according to item 22, which is connectable to capillaries.

[0161] Item 49. The skin substitute according to Item 22, wherein the skin substitute, when transplanted into a recipient subject, is capable of inducing the ingrowth of capillaries of the recipient subject throughout the dermis and down to the basement membrane, particularly, the skin substitute, when transplanted into a recipient subject, is capable of inducing the ingrowth of capillaries of the recipient subject throughout the dermis and down to the basement membrane one week after transplantation into the recipient subject.

[0162] Item 50. The skin substitute according to Item 22, wherein the skin substitute, when transplanted into a recipient subject, is capable of inducing capillary ingrowth in the recipient subject within 4 days after transplantation into the recipient subject.

[0163] Item 51. The skin substitute according to Item 22, wherein the skin substitute, when transplanted into a recipient subject, is capable of exhibiting an oxygen saturation of greater than 50% one week after transplantation into the recipient subject.

[0164] Item 52. The skin substitute according to Item 22, wherein the skin substitute, when transplanted into a recipient subject, is capable of having an oxygenated hemoglobin level that is greater than 30% one week after transplantation into the recipient subject.

[0165] Item 53. The skin substitute according to Item 22, wherein the skin substitute, when transplanted into a recipient subject, is capable of containing a greater number of M2 macrophages than M1 macrophages 3 weeks after transplantation into the recipient subject.

[0166] Item 54. The skin substitute according to Item 22, when transplanted into a recipient subject, is characterized by low or no expression of CK16 and CK17 two weeks after transplantation into the recipient subject, particularly one week after transplantation into the recipient subject.

[0167] Item 55. The skin substitute according to Item 22, wherein all cells of the skin substitute are derived from a single donor subject.

[0168] The present invention is further explained by the following examples and figures, from which further embodiments and advantages can be derived, which are intended to illustrate the invention without limiting its scope. [Brief explanation of the drawings]

[0169] [Figure 1] Figure 1 shows the human CD31 (red) positive endothelial cell fraction after Dynabeads sorting on a cell culture plate. Cell nuclei are stained with Hoechst (blue). Scale bar: 50 μm. [Figure 2] Figure 2 shows the human CD31-negative cell fraction after Dynabeads sorting on a cell culture plate. Cell nuclei are stained with Hoechst (blue). Scale bar: 50 μm. [Figure 3] Figure 3 shows FACS analysis of human BEC and LEC sorted cells (podoplanin is illustratively used as an LEC-specific marker). LEC co-express CD31 (an endothelial-specific marker) and podoplanin, while BEC express CD31 but not podoplanin. [Figure 4] Figure 4 shows human keratinocytes on a cell culture plate. Scale bar: 50 μm. [Figure 5] Figure 5 shows human melanocytes on a cell culture plate. Scale bar: 50 μm. [Figure 6] Figure 6 shows CD146-positive pericytes on a cell culture plate. The entire population of pericytes is CD146-positive, whereas control fibroblasts (FB) show no detectable CD146. CD146+ pericytes are also positive for NG2, but a small proportion are also positive for desmin. CD146+ pericytes are positive for αSMA and CD90, and fibroblasts are also positive for αSMA and CD90. Cell nuclei are stained with Hoechst (blue). Scale bar: 100 μm. [Figure 7]Figure 7 shows whole-mount immunofluorescence staining for CD31 (red) and PROX1 (green) of 3D scaffolds containing sorted and cultured BECs (CD31+PROX1-) / LECs (CD31+PROX1+) and fibroblasts (unstained in this example), demonstrating in vitro capillary tube formation. Scale bar: 50 μm. [Figure 8] Figure 8 shows immunofluorescent staining of angiogenically induced human dermal epidermal skin substitutes one week after transplantation into immunodeficient nude rats. (A) Immunofluorescent co-staining of angiogenically induced skin substitutes for human (hu) CD90 (red) and huCD31 (green). Single staining and overlay are shown. Human CD90 specifically expresses the human dermal compartment of the substitute, while CD31 is expressed only on human endothelial cells. (B) Immunofluorescent co-staining of skin substitutes for huCD31 (red) and huPROX1 (green), visualizing human capillaries (CD31+PROX1-) and lymphatic capillaries (CD31+PROX1+). White arrows indicate double-positive lymphatic microvasculature, while asterisks indicate single-positive capillaries. The inset shows a magnified view of a lymphatic capillary. The white dotted line indicates the junction between the dermis and epidermis. Cell nuclei are stained with Hoechst (blue). The scale bar is 50 μm. [Figure 9] Figure 9 shows the analysis of BEC-LEC-seeded skin substitutes in vivo. (A) Establishment of functional connections (anastomoses) between human and rat CD31-positive capillaries 4 days (4d), 1 week (1w), and 2 weeks (2w) after in vivo implantation. Mean ± SD, n = 5, ***P = 0.0001 (d4), *P = 0.0131 (1w), ns, P = 0.2813 (2w), compared to the non-vascularized control. (B) Quantification of capillary ingrowth (%) in rats 4 days (4d) after implantation. Mean ± SD, n = 5, ***P < 0.0001 (d4), compared to the non-vascularized control. [Figure 10]Figure 10 shows photoacoustic imaging of an animal model. (A-B) BEC-LEC substitutes (white bars) and non-vascularized controls (black bars) were implanted into full-thickness skin defects in nude rats for 1 week (1w). (A) B-mode ultrasound measurements. Oxygen saturation quantification (%). Mean ± SD, n = 5, ***P = 0.0059 compared to non-vascularized controls. (B) Hemoglobin oxygenation quantification (%). Mean ± SD, n = 5, *P < 0.0372 compared to non-vascularized controls. [Figure 11] Figure 11 compares the LEC:BEC ratio in endothelial cells (HDMECs) isolated using two different isolation techniques. HDMECs were isolated from human foreskin using the newly established protocol and compared with the standard procedure (European Patent (EP) No. 3 174 563 B1 and Marino et al., 2014). HDMECs isolated using the old scratch method (European Patent (EP) No. 3 174 563 B1 and Marino et al., 2014) yielded 93.13 ± 24.17% LECs and 6.31 ± 13.09% BECs (n = 48). In contrast, HDMECs isolated using the new isolation method (the present method) yielded 47.92 ± 15.86% LECs (P = 0.0016) and 51.57 ± 16.09% BECs (P = 0.00048) (n = 35). [Figure 12-1] FIG. 12 shows a schematic diagram of the method of the present application. [Figure 12-2] FIG. 12 shows a schematic diagram of the method of the present application. [Figure 13] Figure 13 shows a schematic diagram of the method of Marino et al. (2014) Science Translational Medicine, vol. 6, no. 221. [Figure 14] Figure 14 shows a schematic diagram of the method of Bourland et al. (2018) Scientific Reports, vol. 8, no. 1, p. 13191. [Figure 15] Figure 15 shows a schematic diagram of the method of EP 3 174 563 B1. [Example]

[0170] Example 1: Human dermal endothelial cells were isolated from human skin biopsies and cultured on cell culture plastic in appropriate endothelial cell medium (EGM2, Lonza, Switzerland) at 37°C and 5% CO2. After isolation from the dermis, the cell suspension was incubated with CD31-labeled Dynabeads. CD31-positive cells were sorted from the total cell suspension and then plated onto cell culture plates. CD31-negative cells were also sorted from the total cell suspension and plated onto separate cell culture dishes.

[0171] The purity of the sorted and cultured CD31-positive cells was confirmed by immunofluorescence staining after 5 days of culture (human CD31, clone JC70A, 1:50, Dako). All cells in the culture were positive for CD31 (Figure 1, red). For comparison, all cell nuclei were co-stained with 1 mg / mL Hoechst 33342 (ThermoFischer, Switzerland) (Figure 1, blue).

[0172] Example 2: The CD31-negative sorted cells were also analyzed by immunofluorescence staining (human CD31, clone JC70A, 1:50, Dako), and no CD31 expression was observed in these cultured cells (Figure 2). For comparison, all cell nuclei were stained with Hoechst 33342 (Figure 2, blue).

[0173] Example 3: The sorted and cultured CD31-positive cells were then analyzed using FACS-available antibodies against CD31 and podoplanin (CD31 -The cells were labeled with PE (1:20, clone WM59, BD Biosciences, Switzerland; podoplanin-AF488 clone, 1:50, NC-08, BioLegend, Switzerland) (see Rutsche et al., 2022 and Michalak-Micka et al., 2022 for methods). The labeled cells were used for FACS sorting and were separated into CD31+ / podoplanin+ and CD31+ / podoplanin+ cells (Figure 3). Clearly, two distinct cell fractions, CD31+ / podoplanin+ lymphatic endothelial cells (LEC) and CD31+ / podoplanin+ vascular endothelial cells (BEC), could be gated and sorted separately using the FACS setup (Figure 3).

[0174] Example 4: Keratinocytes and melanocytes can be isolated from the epidermis of human skin samples as described in Michalak-Micka et al., 2022. Confluent monolayers of 2D-cultured keratinocytes exhibit a typical cobblestone morphology under light microscopy (see Figure 4). In contrast, cultured melanocytes exhibit a typical dendritic morphology under light microscopy (see Figure 5).

[0175] Example 5: Human dermal pericytes are a subpopulation of the total CD31-negative cell fraction isolated by Dynabeads sorting. To further separate pericytes from non-pericytes in the total CD31-negative fraction, the CD31-negative fraction was incubated with a CD146 antibody (Biolegend, clone P1H12, 1:50, Switzerland) and the labeled CD146-positive pericytes were sorted (Dynabeads sorting). CD146-positive pericytes and CD146-negative fibroblasts were further cultured separately in vitro on 2D cell culture plates (Figure 6). The difference between fibroblasts and pericytes was demonstrated by the different protein expression patterns using immunofluorescence staining. On cell culture plastic, pericytes express CD146, NG2 (NG2, 1:100, clone LHM2, Novus Biologicals, UK), desmin (Desmin, 1:100, ab8592, Abcam, Germany), CD90 (CD90-FITC, 1:20, clone 5E10, Biolegend, Switzerland), and SMA, whereas fibroblasts do not express CD146, NG2, or desmin, but do express CD90 and SMA (SMA, 1:100, M0851, Dako, Switzerland) (Figure 6). Cell nuclei were stained with Hoechst 33342 (Figure 6, blue).

[0176] Example 6: In vitro-grown angiogenic scaffolds containing human blood and lymphatic capillaries and fibroblasts were implanted into immunodeficient nude rats (see Zimoch et al. for surgical / implantation procedures). After 1 week in vivo, the implanted angiogenic scaffolds were excised and subjected to histological analysis.

[0177] Human CD31-positive (green) endothelial cells reinforcing (lining) the human artificial capillaries were detected by immunofluorescence staining (Figure 8A). These human CD31-positive capillaries were present within the human scaffolds, delineated by human CD90-positive fibroblasts (red), displaying human tissue compared to the host (animal) tissue (Figure 8A). Furthermore, human CD31-positive (red) Prox1-positive (green) lymphatic endothelial cells were shown by immunofluorescence staining compared to CD31-positive (red) non-Prox1-expressing vascular endothelial cells, demonstrating the intermingling of lymphatic and blood capillaries in the human angiogenesis-induced scaffolds in vivo (Figure 8B).

[0178] Example 7: BEC-LEC-containing skin substitutes exhibit enhanced blood perfusion compared with controls without angiogenesis induction The BEC-LEC skin substitutes demonstrate faster blood perfusion than non-vascularized skin substitutes containing only fibroblasts and lacking endothelial cells (Figure 9). As early as 4 days after implantation, the BEC-LEC skin substitutes expressed 25±10% human CD31 + Capillaries are rat CD31 + These perfused capillaries were connected via anastomoses (these perfused capillaries showed rat erythrocytes in their lumen) and contained 75±12% human CD31 + Capillaries are rat CD31 + It was shown that the cells did not connect to capillaries via anastomoses. + In capillary BEC-LEC skin substitutes, the number of perfused capillaries increased to 65±20% at 1 week and 89±10% at 2 weeks.

[0179] Skin substitutes without angiogenesis induction contain 0% human CD31 + Rat CD31 for capillary vessels + Furthermore, the skin substitutes without angiogenesis induction showed 0% rat capillaries at 4 days in vivo, 35±14% rat capillaries at 1 week, and 81±14% rat capillaries at 2 weeks.

[0180] BEC-LEC-containing skin substitutes induce host capillary ingrowth The inventors confirmed that the BEC-LEC skin substitute significantly promoted the ingrowth of rat capillaries into the grafted human dermis. In particular, the BEC-LEC skin substitute showed the presence of rat capillaries 4 days after grafting, whereas the skin substitute without angiogenesis induction showed no rat capillaries 4 days after grafting.

[0181] Importantly, the BEC-LEC skin substitute induced rat capillary ingrowth throughout the dermis down to the basement membrane (just below the epidermis) one week after implantation, whereas the skin substitute without angiogenic induction only showed rat capillaries in the lower dermis one week after implantation.

[0182] Photoacoustic evaluation of oxygenated blood after implantation of BEC-LEC skin substitutes Photoacoustic imaging was performed to quantify in situ oxygen saturation and oxygenated hemoglobin content within the neodermis of the BEC-LEC skin substitutes and controls 1 week after implantation (Figure 10). This approach revealed that the angiogenic BEC-LEC skin substitutes were characterized by significantly higher oxygenation (67±12 vs. 49±4%) and oxygenated hemoglobin content (43±15 vs. 28±3%) compared with controls at 1 week.

[0183] Rapid inflammatory response in angiogenic BEC-LEC skin substitutes after implantation Our data indicate that angiogenesis-induced skin substitutes significantly impact the inflammatory phase during in vivo skin wound healing. In particular, we observed increased numbers of infiltrating monocytes / macrophages and granulocytes in angiogenic human BEC-LEC skin substitutes compared with unangiogenic skin substitutes after implantation in a rat model. This indicates that angiogenic BEC-LEC skin substitutes promote faster and more pronounced wound healing. We further investigated the polarization state of macrophages in these angiogenic BEC-LEC skin substitutes during different stages of wound healing in vivo. We demonstrated that macrophage phenotypes changed from a pro-inflammatory M1 profile 1 week after implantation of angiogenic skin substitutes to an anti-inflammatory, pro-healing M2 phenotype 3 weeks after implantation in vivo. In contrast, skin substitutes without angiogenesis showed a general reduction in the number of macrophages, and in particular a reduction in the number of M2-polarized macrophages, at 1 and 3 weeks in vivo. We speculate that the integration of a mature vascular network into skin substitutes, compared to skin substitutes without angiogenesis induction, orchestrates the fine-tuning of multiple pro- and anti-inflammatory cytokines and growth factors essential at various stages of wound healing.

[0184] Epidermal homeostasis is rapidly achieved after implantation of angiogenic skin substitutes Epidermal keratinocytes express cytokeratins, intermediate filaments of the cytoskeleton. After wounding, the types of cytokeratins differ from those of unwounded epidermal cells. Cytokeratins such as CK1 and CK10 are expressed during homeostasis, whereas cytokeratins such as CK16 and CK17 are present during wound healing. After transplantation of an angiogenic dermal epidermal skin substitute, the wound healing markers CK16 and CK17 are no longer expressed after 1–2 weeks. In contrast, in skin substitutes without angiogenic induction, CK16 and CK17 are still present for 14 days after transplantation and disappear only after 3–4 weeks.

[0185] Example 8: The BEC / LEC isolation method from human skin (referred to herein as the "old method"), as described in European Patent (EP) No. 3 174 563 B1 and by Marino et al., 2014, is based on enzymatic extraction of HDMECs from skin biopsies followed by co-culturing BECs and LECs without separation. Because LECs exhibit significantly higher proliferation potential in vitro, this method results in overgrowth of BECs with LECs. Ultimately, after 2–3 weeks of in vitro culture on tissue culture plastic, the old method results in a nearly pure LEC culture. Therefore, the old method cannot generate sufficient numbers of BECs, making it impossible to generate skin substitutes (with BECs) with a defined number of capillaries, which are important for rapid oxygenation and waste removal in the skin.

[0186] Example 9: Materials and Methods Scaffold Both size and shape depend on the need and availability of cell culture plastic. Cell culture devices can be purchased from commercial suppliers or custom-made. Shapes can be, for example, round (cell culture inserts) or rectangular. Examples include (but are not limited to): Minimum: 24-well cell culture insert size; Maximum: 7 x 8 cm rectangular insert size; an example of a 6 x 6 cm bioprint is shown in Pontiggia et al., 2022.

[0187] Mixing cells with collagen For the angiogenesis-induced hydrogel, 1.5 ml of collagen type I (bovine collagen type I, Symatese, France) was mixed with a 1:1 cell suspension of HDMECs and fibroblasts (total of 100,000 cells / ml), placed in a 6-well insert, and polymerized at 37°C / 5% CO2. After polymerization, EGM-2MV medium was added, and the prepared scaffold was cultured in EGM-2MV medium (Lonza, Basel, Switzerland) for 21 days at 37°C / 5% CO2 to allow capillary tube formation (modified text after Zimoch et al.).

[0188] Isolation of keratinocytes and melanocytes Keratinocytes and melanocytes were isolated as described in Michalak-Micka et al. (2022). Briefly, human skin samples were first cut into small pieces and digested overnight at 4°C in a mixture of 12 U / ml dispase (Corning, New York, USA) and 5 mg / mL gentamicin (Sigma-Aldrich, Buchs, Switzerland) in Dulbecco's phosphate-buffered saline (DPBS, Sigma-Aldrich, Buchs, Switzerland). The epidermis was then mechanically separated from the dermis using forceps. The epidermis was used for the isolation of keratinocytes and melanocytes. To isolate keratinocytes, the epidermis was further digested with 0.5% trypsin-EDTA (Thermo Fisher Scientific, Basel, Switzerland) for 2 minutes at 37°C, after which the cells were resuspended in serum-free keratinocyte medium (CnT-57, CellnTec, Bern, Switzerland) containing 5 μg / mL gentamicin. Melanocytes were isolated from human epidermis by treatment with 0.05% trypsin-EDTA for 5 min at 37°C. Melanocytes were cultured in melanocyte growth medium CnT40 (CellnTec, Bern, Switzerland). The medium was changed every 2 days.

[0189] JPEG2025536605000004.jpg98162

Claims

1. 1. A method for producing a skin substitute, said method comprising the steps of: a. providing a skin tissue sample isolated from a mammalian subject; b. Separating the skin tissue sample into the dermis and epidermis; c. Enzymatically degrading the matrix component of the dermis to obtain a plurality of cells in suspension; d. In a CD31 step, the plurality of cells are subjected to CD31 ＋ Cell fraction and CD31 － sorting the cells into a cell fraction; e. CD31 ＋ The cell fraction was cultured to obtain multiple CD31 ＋ Obtaining the cells; f. In the BEC / LEC step, the plurality of CD31 ＋ sorting the cells into vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs); g. A step of culturing BECs and / or LECs to obtain a skin substitute in a culturing step. The method comprising:

2. The method according to claim 1, wherein the relative amounts of BECs and LECs used in the culturing step are between 70% BECs and 30% LECs and 30% BECs and 70% LECs.

3. 3. The method according to claim 1 or 2, wherein the CD31 step is carried out via magnetic sorting, in particular the CD31 step is carried out via paramagnetic particles.

4. The method according to any one of claims 1 to 3, wherein the BEC / LEC step is performed via fluorescence-based or magnetic sorting, in particular FACS.

5. The method according to any one of claims 1 to 4, wherein in the BEC / LEC step, the BEC-specific marker is selected from the group consisting of PLVAP and PAL-E.

6. The method according to any one of claims 1 to 5, wherein in the BEC / LEC step, the LEC-specific marker is selected from the group consisting of podoplanin, Lyve1, and VEGFR3.

7. After the BEC / LEC process, the BECs are sorted into arterial and venous endothelial cells via arterial and / or venous endothelial cell-specific markers, where the arterial endothelial cell-specific markers are ephrinB2-positive, neuropilin 1 (NRP1), ｈｉｇｈ and the venous endothelial cell-specific marker is selected from the group consisting of ACKR1 (DARC) positivity, neuropilin 2 (NRP2) ｈｉｇｈ The method of any one of claims 1 to 6, wherein the compound is selected from the group consisting of:

8. During the BEC / LEC process, CD146 ＋ Parietal cells are CD31 － The method according to any one of claims 1 to 7, wherein the cell fraction is sorted and added during the culture step.

9. Keratinocytes from the epidermis: by treating the epidermis with a trypsin solution of about 0.5% (w / v); or - by sorting epidermal cells into the MC1R-negative / E-cadherin-positive fraction; The method according to any one of claims 1 to 8, wherein the keratinocytes are isolated and added in a culture step.

10. Melanocytes from the epidermis: by treatment with a trypsin solution of about 0.05% (w / v); or - by sorting epidermal cells into the MC1R-positive / E-cadherin-positive fraction; The method according to any one of claims 1 to 9, wherein the isolated and added melanocytes are cultured.

11. 11. The method according to any one of claims 1 to 10, wherein the digestion of the tissue sample is carried out using an enzyme or a combination of enzymes selected from the group consisting of collagenase II, and / or collagenase IV, and / or a mixture of collagenase I and collagenase II, and / or trypsin.

12. In the culturing step, collagen, and / or fibrin, and / or - gelatin, and / or PEG, and / or - Polyurethane The method according to any one of claims 1 to 11, wherein

13. A skin substitute obtainable by the method according to any one of claims 1 to 12, wherein the dermal portion of the skin substitute contains 35% or more endothelial cells.

14. The skin substitute, when transplanted into a recipient subject, - CD31 in recipient subjects 4 days after transplantation into recipient subjects ＋ It is possible to connect with capillaries, especially CD31 of the donor of the skin substitute. ＋ More than 15% of capillaries were found to be CD31 positive in recipient subjects within 4 days of transplantation into the recipient subject. ＋ capable of connecting to blood capillaries; and / or - capable of inducing capillary ingrowth of a recipient subject throughout the dermis and down to the basement membrane, in particular the skin substitute, when transplanted into a recipient subject, is capable of inducing capillary ingrowth of the recipient subject throughout the dermis and down to the basement membrane one week after transplantation into the recipient subject; and / or - capable of inducing capillary ingrowth in the recipient subject four days after transplantation into the recipient subject; and / or - able to demonstrate an oxygen saturation of greater than 50% one week after transplantation into the recipient subject; and / or - be able to have an oxygenated hemoglobin level of more than 30% one week after transplantation into the recipient subject; and / or - capable of containing a greater number of M2 macrophages than the number of M1 macrophages 3 weeks after transplantation into the recipient subject; and / or - characterized by low or absent expression of CK16 and CK17 at 2 weeks after transplantation into the recipient subject, in particular at 1 week after transplantation into the recipient subject; 14. The skin substitute of claim 13.

15. 15. The skin substitute according to claim 13 or 14, wherein all cells of the skin substitute are derived from a single donor subject, in particular the donor subject is the recipient (autograft).

16. - secondary lymphedema, especially where the skin substitute is cultured from 0% BEC and 100% LEC relative amounts of BEC / LEC; - chronic edema, especially where the skin substitute is cultured from BECs and LECs with a relative amount of BEC / LECs of about 50%; acute burns, especially where the skin substitute is cultured from 100% BECs and 0% LECs in a BEC / LEC relative amount; - arterial ulcers, in particular where the skin substitute is cultured from a BEC / LEC relative amount of 100% BEC and 0% LEC; - venous leg ulcers, especially where the skin substitute is cultured from a BEC / LEC relative amount of 100% BEC and 0% LEC; lymphatic filariasis, in particular where the skin substitutes are cultured from 0% BEC and 100% LEC relative amounts of BEC / LEC; - venous lymphedema, especially where the skin substitute is cultured from a BEC / LEC relative amount of about 50% BEC and about 50% LEC; lymphangioleiomyomatosis, in particular where the skin substitute is cultured from 0% BEC and 100% LEC with a BEC / LEC relative amount; - a skin defect, wherein the skin substitute is cultured from the relative amounts of BEC / LEC as present in the defective skin; 16. The skin substitute of any one of claims 13 to 15 for use in the treatment of a condition selected from the group consisting of:

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