A method for preparing epidermal cell sheets and lyophilisates

Abstract

The present application relates to the field of tissue engineering skin, and specifically discloses a preparation method of epidermal cell membrane and lyophilizate, which comprises the following steps: inoculating isolated human epidermal cells on a human fibroblast feeder layer treated by mitomycin C, culturing to form a layered membrane, then re-contracting the membrane with neutral protease, attaching medical oil gauze, and fixing the isolated membrane with a fixing member to obtain an epidermal cell membrane; and further freezing and drying the isolated membrane to obtain a lyophilizate. The present application can obtain an epidermal cell membrane with layered structure and keratinization characteristics by constructing a feeder layer system and combining mild enzymatic treatment and physical auxiliary separation method, and avoids damage of the membrane. Meanwhile, the problem of long-term storage and transportation of the membrane is solved by lyophilization treatment, and a product for skin repair is provided.

Description

Technical Field

[0001] This invention belongs to the field of tissue-engineered skin, specifically a method for preparing epidermal cell membrane sheets and lyophilized products. Background Technology

[0002] Tissue-engineered skin is an important means of repairing skin defects, and the preparation technology of epidermal cell sheets has received widespread attention in recent years. Epidermal cell sheets refer to continuous, layered sheet-like structures with keratinized characteristics formed by culturing in vitro expanded epidermal cells. They can be directly used for covering and treating skin defects such as burn wounds and chronic ulcers. Traditional methods for constructing epidermal cell sheets typically employ gas-liquid interface culture or use feeder cells to promote epidermal cell stratification. The fibroblast trophoblast layer, in particular, can mimic the dermal microenvironment, secreting various extracellular matrix components and growth factors, which is beneficial for the proliferation and differentiation of epidermal cells.

[0003] After epidermal cell flaps mature, the key technical step in separating them intact from the culture medium is to determine their final quality and clinical feasibility. Currently, commonly used flap separation methods include mechanical scraping and enzymatic digestion. Mechanical scraping uses a cell scraper to directly scrape the flap from the bottom of the culture dish. While simple, it easily leads to edge curling, central rupture, or intercellular separation, resulting in an incomplete flap structure and insufficient area and mechanical strength for subsequent transplantation. Enzymatic digestion typically uses trypsin or neutral protease to detach the flap from the culture medium. However, current techniques often suffer from inadequate control; excessive digestion time leads to over-degradation of intercellular junction proteins, causing the flap to loosen and lose its integrity; insufficient digestion time leaves the flap firmly attached, requiring mechanical tearing, which also easily causes damage. Therefore, developing a method that can gently and completely separate epidermal cell flaps while preserving their layered structure and keratinized characteristics is a pressing technical problem in this field. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing epidermal cell membrane sheets and lyophilized products, thereby resolving the issue in the prior art where improper mechanical scraping or enzyme digestion control during epidermal cell membrane sheet separation leads to membrane breakage and difficulty in obtaining a complete layered structure.

[0005] This invention provides a method for preparing an epidermal cell membrane sheet, comprising the following steps:

[0006] (1) The isolated human epidermal cells were seeded into a culture system containing a human fibroblast trophoblast treated with mitomycin C, wherein the seeding concentration of the human epidermal cells was [missing information]. ;

[0007] (2) Culture for 9-11 days under suitable cell culture conditions until epidermal cell membranes with a layered structure are formed;

[0008] (3) Treat the membrane with a neutral protease solution to retract the membrane from the culture medium bottom, then attach medical gauze to the surface of the membrane, peel off the edge of the membrane, fix the membrane with a fixator, and suspend the membrane in the liquid to complete the membrane separation.

[0009] Preferably, the human fibroblast trophoblast in step (1) is prepared by the following method: human fibroblasts of the 2nd to 5th generation are taken and treated with mitomycin C solution at a concentration of 4 μg / ml for 2 hours, and then the mitomycin C is removed by washing.

[0010] Preferably, the concentration of the neutral protease solution in step (3) is 0.125%, and the treatment time is 10 minutes; the fixing element is a titanium clip, and a total of 6 titanium clips are used to fix it evenly on the edge of the membrane.

[0011] This invention also provides a method for preparing lyophilized epidermal cell membrane sheets, comprising the following steps:

[0012] (1) Prepare and separate epidermal cell membrane sheets according to the above method;

[0013] (2) Freeze the separated epidermal cell membranes at -80°C for 30 minutes;

[0014] (3) Then freeze-dry at 4°C and 10 Pa for 12 hours to obtain freeze-dried epidermal cell membrane sheets.

[0015] Preferably, in step (2), the membrane is placed in a culture dish, sealed with a sealing film, and then frozen. After freezing, the membrane is punctured 5-10 times on the sealing film before being freeze-dried.

[0016] The present invention also provides an epidermal cell membrane sheet, which is prepared by the above method.

[0017] The present invention also provides a freeze-dried epidermal cell membrane sheet, which is prepared by the above method.

[0018] Preferably, the human epidermal cells in step (1) are obtained by the following method: human skin tissue is treated with neutral protease to separate the epidermis, the epidermal tissue is digested with trypsin-EDTA, the cell suspension is collected, and the cells are obtained by filtration, centrifugation and washing.

[0019] Preferably, the human fibroblasts are obtained by the following method: human skin tissue is treated with neutral protease to separate the dermis, the dermal tissue is cut into pieces and digested with type IV collagenase, the cell suspension is collected, and the cells are obtained by filtration, centrifugation and washing.

[0020] Preferably, the filter mesh has a pore size of 70 μm, and the centrifugation conditions are 1300 rpm for 3 minutes; the live cell count is performed using trypan blue staining exclusion method and hemocytometer counting.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] By constructing a human fibroblast trophoblast system treated with mitomycin C and precisely controlling the seeding density and culture period of epidermal cells, the formation of cell sheets with layered structure and keratinization characteristics of epidermal cells in vitro was successfully induced. This method simulates the growth microenvironment of epidermal cells in vivo. The extracellular matrix and growth factors secreted by the trophoblast can effectively promote the adhesion, proliferation and orderly stratification of epidermal cells, making the formed sheet more similar to the natural epidermis in tissue structure and possessing good mechanical strength and operational tolerance.

[0023] Based on this, the present invention uses a specific concentration of neutral protease to gently treat mature membranes, allowing the membranes to naturally retract from the culture medium. This is then combined with medical gauze attachment and titanium clip fixation, achieving complete and non-destructive separation of the membranes and avoiding the damage to cell activity and membrane structure caused by traditional enzyme digestion or mechanical scraping.

[0024] Furthermore, this invention processes the separated epidermal cell membrane sheets using an optimized freeze-drying process, rapidly removing moisture under specific temperature and vacuum conditions to obtain freeze-dried epidermal cell membrane sheets that are easy to store and transport for a long time. These freeze-dried products can quickly restore the membrane morphology and maintain good structural integrity after rehydration, effectively solving the problems of difficulty in long-term preservation of freshly prepared cell membrane sheets, time-consuming on-site preparation, and poor batch stability. This provides a ready-to-use tissue engineering product for skin defect repair, burn wound coverage, and transdermal drug absorption research.

[0025] The entire preparation process is standardized and reproducible, and the resulting product does not require complex cryopreservation and thawing procedures, showing promising prospects for clinical translation and industrialization. Attached Figure Description

[0026] Figure 1 This is a complete process flow diagram of the present invention for obtaining the final epidermal cell membrane sheet and lyophilized product from skin tissue;

[0027] Figure 2 This is a partial experimental process diagram of the present invention, specifically including:

[0028] Figure 2 A shows the cell fusion diagram (left) and the cell membrane formation diagram (right);

[0029] Figure 2 B shows the membrane separation diagram at the bottom of the dish (left) and the membrane oil yarn fixation diagram (right).

[0030] Figure 2 C is an HE staining image of epidermal cell membranes;

[0031] Figure 2 D is a scanning electron microscope image of an epidermal cell membrane sheet;

[0032] Figure 3 This is a diagram of the freeze-dried film of the present invention;

[0033] Figure 4 This is a diagram showing the rehydration of the membrane in this invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1: Isolation and culture of human epidermal cells and dermal fibroblasts, such as Figure 1 As shown

[0036] 1.1 Materials and Reagents

[0037] Skin tissue source: Sterile foreskin tissue obtained from healthy male donors under the age of 30 after circumcision; informed consent from the donor and tissue collection in accordance with medical ethics.

[0038] Triple-antibiotic PBS solution: Three antibiotics are added to sterile phosphate-buffered saline (PBS, pH 7.2-7.4): penicillin (100 U / ml), streptomycin (100 μg / ml), and amphotericin B (0.25 μg / ml). This solution is used for tissue rinsing and cell washing to prevent bacterial and fungal contamination.

[0039] Neutral protease solution: Weigh neutral protease (Dispase II, activity ≥0.5 U / mg) and dissolve it in sterile PBS to prepare a 0.125% (w / v) solution. Filter the solution through a 0.22 μm filter membrane for sterilization and store at 4℃ for later use.

[0040] Type IV collagenase solution: Dissolve type IV collagenase (Sigma-Aldrich, catalog number C5138, activity ≥125 U / mg) in Hanks balanced salt solution (HBSS, free of sodium chloride). , Prepare a solution of 1500 U / ml in the solution, filter it through a 0.22 μm filter membrane for sterilization, and prepare it fresh before use.

[0041] Rat tail collagen: Prepared using conventional acid extraction methods in this field; specifically: SPF-grade rat tail tendons were soaked in 0.1% acetic acid solution at 4°C for 48 hours, centrifuged, and the supernatant was collected, dialyzed, and then lyophilized for storage. Before use, it was diluted to 30 μg / ml with 0.1% acetic acid and filtered through a 0.22 μm filter membrane; alternatively, commercially available rat tail collagen type I (such as Corning, catalog number 354236) can be purchased directly.

[0042] XR keratinocyte culture medium: serum-free, low calcium ion (0.06 mM) For keratinocyte-specific culture media, commercially available products such as Gibco™ Epilife® medium (catalog number MEPI500CA) or PromoCell® Keratinocyte Growth Medium 2 (catalog number C-20011) can be used, along with complementary growth additives (containing bovine pituitary extract, epidermal growth factor, insulin, etc.).

[0043] DMEM high glucose medium: Dulbecco's Modified Eagle Medium, containing 4.5 g / L glucose, L-glutamine, sodium pyruvate, and free of HEPES; add fetal bovine serum (FBS) to a final concentration of 10% or 20% as needed for the experiment.

[0044] Trypsin-EDTA solution: 0.25% (w / v) trypsin, containing 0.02% (w / v) EDTA·4Na, soluble in anhydrous water. , Sterilize by filtration through a 0.22 μm filter membrane in Hanks' solution or PBS.

[0045] Mitomycin C solution: Dissolve mitomycin C powder in sterile Hanks solution to prepare a 4 μg / ml stock solution. Sterilize by filtration through a 0.22 μm filter membrane. Store at 4°C protected from light and use within one week.

[0046] Protease terminating agent: DMEM high glucose medium containing 10% (v / v) fetal bovine serum (FBS); components such as α-macroglobulin in FBS can effectively neutralize the activity of neutral proteases; PBS solution containing 0.5 mg / ml soybean trypsin inhibitor can also be used.

[0047] Medical oil gauze: Mansalin oil gauze (i.e., petroleum jelly gauze) is a sterile gauze impregnated with yellow or white petroleum jelly, with a size of 5 cm × 5 cm or 10 cm × 10 cm, used to support and transfer cell membranes.

[0048] Titanium clip: Made of medical-grade pure titanium, sterile packaged, measuring 2 mm × 3 mm, used to fix the edge of the membrane.

[0049] 1.2 Separation of epidermis and dermis

[0050] Take sterile foreskin tissue and place it in a 50 ml centrifuge tube. Immerse the tissue in povidone-iodine solution and disinfect it three times for three minutes each time. Then transfer the tissue to a 6 cm sterile culture dish and rinse it three times with 5 ml of PBS solution containing triple antibodies each time. Under a dissecting microscope, use sterile ophthalmic scissors and forceps to trim and remove subcutaneous connective tissue, fat and blood vessels until the porcelain white and shiny dermal tissue layer is exposed.

[0051] Cut the trimmed tissue into strips 3-5 mm wide, and rinse three times with triple antibody PBS. Transfer the tissue strips to 15 ml centrifuge tubes, add 0.125% neutral protease solution, ensuring the liquid completely submerges the tissue strips, mix thoroughly, and incubate overnight (12-18 hours) at 4°C.

[0052] The following day, the tissue strips were transferred to a 6 cm culture dish and rinsed three times with triple-antibody PBS. Under a dissecting microscope, the epidermis and dermis were gently separated using two fine forceps: one forceps held the dermal side, and the other forceps held the edge of the epidermis, and the intact epidermal layer was peeled off from the dermis with a gentle tearing motion. The separated epidermis was transferred to a 6 cm culture dish containing 2 ml of triple-antibody PBS, and the dermis was transferred to a 10 cm culture dish containing triple-antibody PBS for later use.

[0053] 1.3 Epidermal cell culture

[0054] Coat T75 cell culture flasks with 30 μg / ml rat tail collagen solution: Add 5 ml of collagen solution to each flask, gently shake to evenly cover the bottom of the flask, and let stand at room temperature for 30 minutes; remove the collagen solution, rinse twice with PBS, and place in a clean bench to air dry for later use.

[0055] Remove the triple antibody PBS from the culture dish containing the epidermis, add 3 ml of 0.25% trypsin-EDTA solution preheated to 37°C, and repeatedly pipette the epidermal tissue block for 2 minutes using a 1 ml pipette. After the suspension becomes turbid and the epidermal tissue block becomes transparent, transfer the digestion solution to a 50 ml centrifuge tube containing 15 ml of DMEM high-glucose medium with 10% FBS to stop the digestion. Repeat the above digestion operation 3-4 times until the epidermal tissue block is basically digested.

[0056] Filter the cell suspension in the centrifuge tube through a 70 μm pore size cell filter, collect the filtrate, and centrifuge at 1300 rpm (approximately 250 × g) for 3 minutes; aspirate the supernatant, add 10 ml of triple antibody PBS to resuspend the cells, and centrifuge again at 1300 rpm for 3 minutes to wash; aspirate the supernatant, add 10 ml of XR keratinocyte culture medium to resuspend the cells; take a small amount of cell suspension for counting.

[0057] Cell counting was performed using a hemocytometer combined with trypan blue staining exclusion method: 20 μl of cell suspension was mixed with 20 μl of 0.4% trypan blue solution and stained at room temperature for 2 minutes. 10 μl of the mixture was added to the hemocytometer, and live cells (unstained) and dead cells (blue) were counted under a microscope. Cell density and viability were calculated according to the formula.

[0058] Adjust cell density to The cells were inoculated into T75 culture flasks pre-coated with rat tail collagen, with 10 ml of cell suspension in each flask. (1 cell / flask); one adult foreskin can be used to inoculate 4 flasks of T75; place the culture flask in Culture in a 37℃ cell culture incubator; replace with fresh XR keratinocyte culture medium every other day; when the cells reach 80%-90% confluence, passage at a 1:2 ratio.

[0059] 1.4 Dermal cell (fibroblast) culture

[0060] The separated dermal tissue was rinsed twice with triple-antibody PBS, and then cut into approximately [size missing] pieces using ophthalmic scissors. Remove small fragments; add 10 ml of DMEM high-glucose medium containing 20% ​​FBS, and transfer the tissue suspension to a 50 ml centrifuge tube; add 5 ml of freshly prepared 1500 U / ml type IV collagenase solution (medium to collagenase volume ratio 2:1), and mix thoroughly; place the centrifuge tube in a 37°C cell culture incubator, and shake for 30 seconds every 15 minutes, for a total digestion time of 1.5 hours; alternatively, digestion can be performed using a 37°C constant temperature shaker (120 rpm).

[0061] After digestion, the cell suspension was filtered through a 70 μm cell filter, and the filtrate was collected and centrifuged at 1300 rpm for 3 minutes. The supernatant was removed, and the cells were resuspended in 10 ml of DMEM high-glucose medium containing 10% FBS. After counting, the cell density was adjusted to [a specific concentration]. Inoculate 10 ml into each T75 culture flask; place in... Culture in a 37℃ incubator; change the culture medium every other day; after the cells reach 90% confluence, passage them at a 1:2 ratio, and use cells from passages 2-5 for subsequent experiments.

[0062] Example 2: Preparation of human fibroblast trophoblast

[0063] Human dermal fibroblasts from passages 2-5 were collected. The culture medium in the culture flask was aspirated, and the cells were gently washed with 10 ml of triple antibody PBS and then aspirated. 3 ml of 0.25% trypsin-EDTA solution was added, the flask was capped, and the flask was placed in a 37°C incubator for 90 seconds to digest (observe under a microscope that the cells become round and partially detach). Immediately, 3 ml of DMEM culture medium containing 10% FBS was added to stop the digestion. The cell suspension was transferred to a 50 ml centrifuge tube and centrifuged at 1300 rpm for 3 minutes. The supernatant was aspirated, and the cells were resuspended in 10 ml of triple antibody PBS and washed again by centrifugation.

[0064] Aspirate the supernatant, resuspend the cells in DMEM high-glucose medium containing 10% FBS, and adjust the cell density to [the desired level]. Seed the cells into 6 cm cell culture dishes, adding 3.5 ml of cell suspension to each dish; change the culture medium every other day; when the cells have grown to 70%-80% confluence of the bottom area of ​​the culture dish, aspirate the culture medium and wash once with triple antibody PBS; add 2.5 ml of 4 μg / ml mitomycin C solution prepared with Hanks' solution, cover the culture dish, and incubate at 37°C for 2 hours.

[0065] After incubation, remove the mitomycin C solution, add 3 ml of triple antibody PBS, soak for 3 minutes, gently rinse and remove; repeat washing 5 times (the PBS added in the 5th time is removed when inoculating epidermal cells later); the fibroblasts treated with mitomycin C are the feeder cells, which can be used immediately or stored in PBS at 4°C for no more than 2 hours.

[0066] Example 3: Culture of human epidermal cell membrane sheets

[0067] Human epidermal cells from passages 2-5 were digested, centrifuged, and washed according to the method in Example 1.3, and then resuspended in epidermal cell sheet culture medium. This medium consisted of a mixture of XR keratinocyte culture medium (90%, v / v) and DMEM high-glucose medium containing 10% FBS (10%, v / v). The cell density was adjusted to 5 × 10⁶ cells / year. 6 / ml.

[0068] Remove the PBS from the last wash in the 6 cm culture dish containing the human fibroblast trophoblast layer prepared in Example 2; inoculate each dish with 3.5 ml (approximately) of the above cell suspension. (1 epidermal cell / dish); gently shake the culture dish to distribute the cells evenly; place in 5%... Incubate in a 37℃ incubator.

[0069] Change the culture medium daily: carefully aspirate the old medium and slowly add 3.5 ml of fresh, preheated epidermal cell sheet culture medium along the side of the dish; closely observe cell morphology changes during culture; after about 3-4 days, cells will aggregate into small colonies; after 7-8 days, cells will gradually fuse to form a continuous layer; after 9-11 days, cells will grow in layers, with blurred cell boundaries and highly refractive keratin-like protein granules appearing on the surface, indicating that the sheet has formed to a suitable thickness, as shown in the following figures. Figure 2 A.

[0070] Example 4: Isolation of human epidermal cell membranes

[0071] After 9-11 days of epidermal cell seeding and culture, membrane separation was performed in a clean bench. The culture medium was aspirated from the culture dish, and 5 ml of triple antibody PBS was added to gently rinse once and then aspirated. 2.5 ml of 0.125% neutral protease solution was added to cover the entire membrane, and the culture dish was placed in a 37°C incubator for 10 minutes. During this period, the dish could be removed for observation: obvious retraction and curling were visible around the periphery of the membrane, indicating separation from the bottom of the culture dish.

[0072] After incubation for 10 minutes, aspirate the neutral protease solution and immediately add 2.5 ml of protease stop agent (DMEM high glucose medium containing 10% FBS). Stop digestion for about 2 minutes at room temperature. Aspirate the liquid and wash 3 times with triple antibody PBS, 5 ml each time.

[0073] Place a piece of cut, equal-area Mansalex oil gauze (Vaseline gauze) into a culture dish and attach it completely to the surface of the epidermal cell membrane. Press gently to ensure a tight fit. Use sterile forceps to gently peel the membrane from the edge, separating the edge of the membrane from the bottom of the culture dish, leaving space for inserting titanium clips. Take 6 medical titanium clips and fix them evenly at the 3 o'clock, 6 o'clock, 9 o'clock, 12 o'clock and center positions on the edge of the membrane, clamping the edge of the membrane about 1-2 mm wide at each point.

[0074] Hold the titanium clip with tweezers and gently lift it upwards to completely separate the epidermal cell membrane sheet along with the gauze from the bottom of the culture dish. Add 2 ml of triple antibody PBS to the culture dish. The membrane sheet will be seen to be suspended in the PBS, completing the separation and removal. If not used immediately, the separated membrane sheet along with the gauze can be placed in DMEM medium and stored in a 37°C incubator for no more than 3 hours.

[0075] Example 5: Preparation of Lyophilized Epidermal Cell Membrane Patches

[0076] Specifically, such as Figure 2 B. Transfer the epidermal cell membrane sheet (along with the gauze and titanium clip) separated and peeled off in Example 4 to a new sterile culture dish and aspirate the PBS solution; add 3 ml of triple antibody PBS and gently rinse twice, then aspirate the PBS; remove the culture dish lid and seal the entire culture dish tightly with sealing film.

[0077] Immediately place the sealed petri dish in a -80°C freezer for 30 minutes. Remove the petri dish and use a sterile syringe to puncture 5-10 small holes in the sealing film to ensure that water vapor can escape during the freeze-drying process. Place the petri dish in a freeze dryer, set the temperature to 4°C and the vacuum degree to 10 Pa (absolute pressure), and freeze-dry for 12 hours.

[0078] After freeze-drying, the culture dish was removed, yielding freeze-dried epidermal cell membrane sheets. These freeze-dried sheets exhibited a dry, sheet-like structure with a visible white to pale yellow keratin membrane on the surface. (Specific details are as follows...) Figure 3 The freeze-dried product, along with the culture dish, can be sealed and stored in a 4°C refrigerator for up to 6 months or more.

[0079] Example 6: Comparison of different generations

[0080] Following the methods in Examples 1-3, epidermal cells and fibroblasts from the 2nd, 3rd, 4th, and 5th generations were used for trophoblast preparation and membrane culture. The results showed that cells from the 2nd to 5th generations could form epidermal cell membranes with complete structure and uniform thickness. Among them, the membranes formed by epidermal cells from the 3rd and 4th generations had a higher degree of keratinization and better mechanical strength.

[0081] Example 7 Comparison of different freeze-drying parameters

[0082] Following the method in Example 5, the effects of freezing temperature (-80℃, -40℃, -20℃), freezing time (15 min, 30 min, 60 min), freeze-drying temperature (-20℃, 4℃, 25℃), and freeze-drying time (6 h, 12 h, 24 h) on membrane integrity were investigated. The results showed that the combination of freezing at -80℃ for 30 minutes and freeze-drying at 4℃ for 12 hours could maintain the original morphology and structural integrity of the membrane to the greatest extent. Too high a temperature or too short a time would lead to membrane breakage or loss of cell activity after rehydration.

[0083] IV. Cell Counting and Viability Detection Methods

[0084] In this invention, both cell counting and live cell counting are performed using a hemocytometer combined with trypan blue staining for exclusion; the specific procedures are as follows:

[0085] Mix 10 μl of cell suspension with 10 μl of 0.4% (w / v) trypan blue solution (dissolved in PBS) and stain at room temperature for 2-3 minutes.

[0086] Take 10 μl of the stained mixture and add it to the counting chamber of the hemocytometer. Gently cover with a coverslip to avoid generating air bubbles.

[0087] Under an inverted microscope, count the live cells (bright, unstained) and dead cells (stained blue) in four large squares (each large square contains 16 small squares).

[0088] Calculate using the following formula:

[0089]

[0090]

[0091] V. Product Performance Testing

[0092] 5.1 Observation of membrane morphology

[0093] The epidermal cell membrane obtained in Example 3 was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Microscopic examination revealed that the membrane consisted of 3-5 layers of epidermal cells with tight intercellular connections and a keratinized layer on the surface. Scanning electron microscopy showed that the membrane surface was smooth, intact, and without cracks. Specifically... Figure 2 C and Figure 2 D.

[0094] 5.2 Rehydration test of freeze-dried products

[0095] Take the lyophilized product obtained in Example 5, add 5 ml of sterile PBS, and let it stand at room temperature for 5 minutes; it can be seen that the lyophilized product quickly absorbs water and rehydrates, restoring the membrane morphology, as shown in the following figure. Figure 4 The rehydrated membranes retained their intact sheet-like structure without breakage; HE staining showed that the cell morphology after rehydration was not significantly different from that before freeze-drying.

[0096] The embodiments of the present invention are given for the purposes of illustration and description. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made by those skilled in the art to the above embodiments within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an epidermal cell membrane sheet, characterized in that, Includes the following steps: (1) The isolated human epidermal cells were seeded into a culture system containing a human fibroblast trophoblast treated with mitomycin C, wherein the seeding concentration of the human epidermal cells was [missing information]. ; (2) Culture for 9-11 days under suitable cell culture conditions until epidermal cell membranes with a layered structure are formed; (3) Treat the membrane with a neutral protease solution to retract the membrane from the culture medium bottom, then attach medical gauze to the surface of the membrane, peel off the edge of the membrane, fix the membrane with a fixator, and suspend the membrane in the liquid to complete the membrane separation.

2. The method according to claim 1, characterized in that, The human fibroblast trophoblast described in step (1) is prepared as follows: human fibroblasts of the 2nd to 5th generation are taken and treated with mitomycin C solution at a concentration of 4 μg / ml for 2 hours, and then washed to remove mitomycin C.

3. The method according to claim 1, characterized in that, The concentration of the neutral protease solution in step (3) is 0.125%, and the treatment time is 10 minutes; the fixing element is a titanium clip, and a total of 6 titanium clips are used to fix it evenly on the edge of the membrane.

4. A method for preparing a freeze-dried epidermal cell membrane sheet, characterized in that, Includes the following steps: (1) Prepare and separate epidermal cell membrane sheets according to any one of claims 1-3; (2) Freeze the separated epidermal cell membranes at -80°C for 30 minutes; (3) Then freeze-dry at 4°C and 10 Pa for 12 hours to obtain freeze-dried epidermal cell membrane sheets.

5. The method according to claim 4, characterized in that, In step (2), the membrane is placed in a culture dish, sealed with a sealing film, and then frozen. After freezing, the membrane is punctured 5-10 times on the sealing film before being freeze-dried.

6. An epidermal cell membrane sheet, characterized in that, It is prepared by the method described in any one of claims 1-3.

7. A freeze-dried epidermal cell membrane sheet, characterized in that, Prepared by the method described in claim 4 or 5.

8. The method according to claim 1, characterized in that, The human epidermal cells mentioned in step (1) are obtained by the following method: human skin tissue is treated with neutral protease to separate the epidermis, the epidermal tissue is digested with trypsin-EDTA, the cell suspension is collected, and the cells are obtained by filtration, centrifugation and washing.

9. The method according to claim 1, characterized in that, The human fibroblasts were obtained by the following method: human skin tissue was treated with neutral protease to separate the dermis, the dermal tissue was cut into pieces and digested with type IV collagenase, the cell suspension was collected, filtered through a filter screen, and washed by centrifugation.

10. The method according to claim 8 or 9, characterized in that, The filter mesh has a pore size of 70 μm, and the centrifugation conditions are 1300 rpm for 3 minutes; live cell counting is performed using trypan blue staining exclusion method and hemocytometer counting.

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