A frozen microneedle for transdermally delivering living stem cells and a preparation method and application thereof

Abstract

This invention discloses a cryo-microneedle for transdermal delivery of living stem cells, its preparation method, and its application. The preparation steps include: 1. Preparing a foam-like methacrylated gelatin hydrogel precursor; 2. Cryo-hydrogel synthesis: The foam-like methacrylated gelatin hydrogel precursor is mixed with buffer A containing a photoinitiator, heated and dissolved to obtain the methacrylated gelatin hydrogel, and then CryoStor CS10 cell cryopreservation solution is added to obtain the cryo-hydrogel; 3. Preparing the cryo-microneedle: (1) cryo-needle tip perfusion, (2) cryo-base filling, (3) programmed cooling freezing, demolding, and obtaining the cryo-microneedle. The cryo-microneedle of this invention can quickly and effectively penetrate the skin, precisely delivering various stem cells or organoids to the dermis in a minimally invasive and painless manner; the segmented cryo-microneedle has a pure liquid base that melts rapidly without causing permanent damage to the skin.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, specifically to a cryo-microneedle for transdermal delivery of living stem cells, its preparation method, and its application. Background Technology

[0002] Generally, mature human organs and tissues have limited regenerative capacity after atrophy or necrosis. While the skin, as the largest organ in the human body, can repair itself after trauma, adult skin has limited recovery capabilities. Unlike infant skin, adult skin's self-repair typically results in scar tissue. Although scar formation fulfills basic functions such as preventing infection and dehydration, it usually hinders the full recovery of skin function and prevents the regeneration of skin appendages such as hair follicles, sweat glands, and sebaceous glands. Hair follicle regeneration is a hallmark of optimal skin repair. Adult skin scarring and hair follicle damage can lead to permanent hair loss. Hair loss refers to the absence or loss of hair in localized or diffuse areas. Androgenetic alopecia (AGA) is the most common non-scarring form of alopecia, causing damage to the skin's inherent functions and significantly impacting patients' mental health and quality of life. Unfortunately, conventional treatments for AGA do not increase the source of hair follicles; therefore, there is an urgent need to develop new methods for regenerating and reconstructing hair follicles.

[0003] To advance hair follicle replacement therapy, scientists are combining principles of engineering and life sciences to develop biological alternatives that can restore, maintain, or improve tissue function. Tissue engineering methods, particularly for hair follicle reconstruction, show great promise. Classic rodent hair follicle reconstruction methods, such as injection, chamber, and flap techniques, have successfully reconstructed hair. In the injection method, cells are mixed in a syringe and injected into the subcutaneous layer of the skin, where they form cystic structures of varying sizes depending on the injection volume. The chamber method involves creating a full-thickness skin defect in vivo, placing a silicone cavity within the wound, and then injecting the cell mixture into the cavity, allowing the defective skin and hair follicles to regenerate. Similarly, the flap method requires separating a full-thickness pedicled flap from the skin, then placing a sheet of intact epidermal skin covered with dermal cells onto a sterile silicone membrane, implanting the silicone membrane under the flap, and sealing the wound with tissue adhesive. All three classic hair follicle reconstruction methods can produce newly generated hair follicles derived from cell differentiation, which is beneficial for analyzing intercellular signaling interactions and developmental biology research. However, all three methods have some limitations. Almost all hair follicles reconstructed through patch experiments are implanted in anatomically abnormal locations, thus failing to break through the skin surface to form physiologically guided follicles. While the follicles produced by the chamber method are morphologically similar to natural follicles, it requires a maximum number of cells and creates invasive wounds, resulting in the disadvantages of infection risk and low tolerance. Similarly, flap experiments require the use of intact epidermal flaps and surgical intervention, carrying risks of damage and infection. Furthermore, implanting silicone flaps can cause foreign body inflammation. Finally, among the three classic high-frequency reconstruction methods, the number and location of reconstructed hairs are difficult to control.

[0004] Microneedling is an emerging, micrometer-scale, minimally invasive delivery method. Using microneedles, targeted drugs or small molecules can be delivered painlessly to specific layers of skin and other tissues by creating temporary microchannels within the stratum corneum. Microneedles, being micrometers in size, cannot reach pain receptors deep in the dermis, so they cause less pain than subcutaneous injection needles. However, the manufacture of microneedles requires the use of specific materials and processes to ensure sufficient mechanical strength for successful percutaneous puncture. Microneedles have been successfully used to deliver substances such as drugs, metal ions, gases, and nanoparticles. This technology offers a potentially novel strategy for tissue and organ reconstruction.

[0005] However, in traditional microneedle fabrication processes, when using hydrogels as the material, a heat-drying process is required to obtain microneedles with sufficient mechanical strength to penetrate the stratum corneum. This process typically involves drying at room temperature for 24-48 hours or at 30-35°C for 5-6 hours. During drying, the water in the hydrogel evaporates, increasing its concentration and making the hydrogel network denser, thereby enhancing the mechanical strength of the microneedles. However, stem cell activity is highly sensitive to the surrounding environment. Heat drying, under unsuitable temperatures and conditions of gradually depleting moisture and nutrients, makes it difficult for stem cells to maintain good activity. Therefore, microneedles carrying living organic matter using traditional drying methods have not yet been developed, and microneedles that can maintain high mechanical strength while preserving the vitality and stemness of living cells have not yet been developed.

[0006] Organoids are models built using 3D in vitro cell culture systems that closely resemble their in vivo source tissues or organs. These 3D in vitro culture systems can replicate the complex spatial morphology of differentiated tissues and exhibit interactions and spatial positioning between cells and between cells and their surrounding matrix. Furthermore, they can demonstrate physiological responses similar to differentiated tissues and organs in vivo, exhibiting a very high degree of similarity to their source tissues. The construction of organoid models, using cells derived from specific germ layers of tissues and centrifuged and compressed into the extracellular matrix to create layered three-dimensional co-culture models, has been extensively studied in the oral cavity, intestinal tract, and respiratory systems, and has also received considerable attention in the field of hair follicle tissue engineering and regeneration. Using skin-derived cells to construct organoids and then implanting them can successfully reconstruct hair follicles, potentially expanding the source of hair follicles. However, the in vivo implantation of hair follicle organoids faces many challenges, such as the inability to form hair follicles in one step, the need for invasive surgical trauma, and the complexity of model construction and implantation procedures. To overcome these problems, a convenient, minimally invasive, efficient delivery method that can maintain cell viability is needed. Summary of the Invention

[0007] The purpose of this invention is to address the above-mentioned problems by providing a method for preparing cryo-microneedles for transdermal delivery of living stem cells, comprising the following steps:

[0008] I. Preparation of foam-like methacrylated gelatin hydrogel precursor;

[0009] II. Synthesis of cryo-hydrogels:

[0010] Take the foam-like methacrylated gelatin hydrogel precursor prepared in step one, add buffer A containing a photoinitiator, heat to dissolve, and obtain a methacrylated gelatin hydrogel with a concentration of 80-120 mg / ml; take the methacrylated gelatin hydrogel, add CryoStor CS10 cell cryopreservation solution, mix well, sterilize, and obtain a sterile cryogel; the volume ratio of methacrylated gelatin hydrogel to CryoStor CS10 is 1:0.8-1.2;

[0011] III. Preparation of cryo-microneedles, including the following steps:

[0012] (1) Cryo-needle tip perfusion: Centrifuge the prepared solution containing live stem cells and discard the supernatant. Resuspend the cells in the prepared cryo-hydrogel. Inject the cryo-hydrogel containing live cells into the needle cavity of a sterile microneedle mold. Then centrifuge the microneedle mold to remove the air trapped in the needle tip and make the cells in the suspension densely packed at the tip of the mold needle.

[0013] (2) Filling the cryogenic substrate: Perform the following operations at 4-8°C: remove excess hydrogel from the microneedle mold, irradiate the microneedle tip with ultraviolet light to crosslink the raw materials, then add the cell cryopreservation solution CryoStor CS2 to the microneedle mold to fill the substrate, and then centrifuge to remove the air trapped in the needle tip.

[0014] (3) Programmed cooling and freezing: The mold is subjected to programmed cooling and freezing, and then demolded to obtain frozen microneedles.

[0015] The specific method of programmed cooling and freezing in step (3) is as follows: freeze the mold at -20℃ to -30℃ for 2 to 4 hours, remove the frozen microneedles from the mold at low temperature, then freeze at -70℃ to -90℃ for 2 to 4 hours, and finally freeze in liquid nitrogen for 1 to 3 hours to obtain frozen microneedles.

[0016] In step one, a foam-like methacrylated gelatin hydrogel precursor is prepared using methacrylic anhydride and gelatin as raw materials.

[0017] The gelatin is selected from pig skin gelatin powder, fish skin gelatin powder, fish scale gelatin powder, cow skin gelatin powder, cow bone gelatin powder, and chicken skin gelatin powder; the ratio of methacrylic anhydride to gelatin is 5-7 mL:10 g, preferably 6 mL:10 g.

[0018] 4. The preparation method according to claim 1, characterized in that: the photoinitiator in step two is selected from lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, 2,2-dimethoxy-2-phenylphenylphenylethyl, Irgacure-2959, Irgacure-184, Irgacure-907, and the buffer solution A is a phosphate buffer, a sodium bicarbonate buffer, or a sodium citrate buffer;

[0019] The concentration of the methacrylated gelatin hydrogel is 90-110 mg / ml, of which the concentration of the photoinitiator is 0.8-1.2 mg / ml; the ratio of the methacrylated gelatin hydrogel to CryoStor CS10 is 1:0.9-1.1 by volume.

[0020] In step three

[0021] In step (2) of freezing the substrate filling, the raw material is cross-linked by irradiating the tip of the microneedle with ultraviolet light for 12 to 60 seconds.

[0022] The specific steps of step (3) of programmed cooling are as follows: the mold is frozen at -20°C for 2 hours, the frozen microneedles are gently peeled off the mold on dry ice and placed in a sterile container, then frozen at -80°C for 2 hours, and then frozen in liquid nitrogen at -196°C for 1 hour to obtain the frozen microneedles.

[0023] The bottom diameter of each needle in the microneedle mold is 600-800μm, the height is 1000-1200μm, and the distance between needle tips is 1500-2500μm.

[0024] The living stem cells include hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, liver stem cells, muscle satellite cells, skin stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, and embryonic stem cells; the cell content in the cryogel containing living stem cells is 0.8–1.4 × 10⁻⁶ cells / year. 6 mL -1 ;

[0025] Preferably, the living stem cells are skin stem cells, which include epidermal stem cells and dermal stem cells, wherein the cell ratio of epidermal stem cells to dermal stem cells is 1-9:1-9, preferably 1-5:1-5, 1-3:1-3, or 1-2:1-2;

[0026] Another object of the present invention is to provide a cryo-microneedle for transdermal delivery of living stem cells, prepared using any of the preparation methods described above.

[0027] A final object of the present invention is to provide the application of the above-described cryo-microneedles in the preparation of microneedles for transdermal delivery of living stem cells.

[0028] Preferably, in the above application technology solution, the cryo-microneedles are loaded with skin stem cells for the purpose of reconstructing hair follicles to treat hair loss.

[0029] This invention draws inspiration from the physical alteration of water freezing in the natural environment. By employing cryogenic freezing, it achieves enhanced mechanical strength while avoiding the harmful heat drying process used in stem cell preparation. However, simply freezing water into ice to carry live cells presents problems because ice crystals can damage cells during the freeze-thaw process. While ice crystal formation is unavoidable, cryopreservation solutions can enhance the low-temperature tolerance of the solution and reduce damage to frozen cells. This invention uses a cryopreservation solution containing dimethyl sulfoxide (DMSO) to prepare microneedles. During cell freezing, DMSO increases the porosity of the cell membrane, allowing water to flow more freely through it. Furthermore, DMSO can increase intracellular solute concentration, preventing the formation of crystallites and thus aiding in the vitrification of water at low temperatures, reducing cell damage. This achieves the goal of loading and delivering live cells onto microneedles.

[0030] The beneficial effects of this invention are:

[0031] In this invention, we constructed segmented cryo-microneedles carrying organoids for hair follicle reconstruction. By optimizing cell cryopreservation solutions and methacrylated gelatin, we synthesized a cryogel using a cell cryopreservation solution containing dimethyl sulfoxide and methacrylated gelatin. This cryogel is suitable for both microneedle preparation and maintaining stem cell survival. Microneedles prepared from the cryogel possess strong mechanical strength, enabling rapid and effective skin penetration to precisely deliver organoids to the dermis in a minimally invasive and painless manner. The segmented cryo-microneedles, with their purely liquid base, melt rapidly without causing permanent skin damage. The hair follicle organoids carried by the cryo-microneedles undergo a series of developmental processes within the skin, ultimately reconstructing mature hair follicles. The reconstructed hair can biomimeticly penetrate the skin surface and maintain structural and functional integrity, stably reconstructing structures such as dermal papilla cells, sympathetic nerves, and arrector pili muscles. Attached Figure Description

[0032] Figure 1 These are Fourier transform infrared spectra of frozen hydrogel and methacrylated gelatin hydrogel.

[0033] Figure 2 This is a flowchart of the preparation method of the cryo-microneedles of the present invention.

[0034] Figure 3 This is a schematic diagram of the structure of the cryo-microneedle of the present invention.

[0035] Figure 4 This is a physical image of the cryo-microneedle of the present invention.

[0036] Figure 5 These are scanning electron microscope images of cryogenically prepared microneedles and conventionally prepared microneedles using heat drying, based on the present invention.

[0037] Figure 6 This invention describes the room temperature melting process of cryogenic microneedles.

[0038] Figure 7 This is the compressed form of the cryo-microneedles of the present invention.

[0039] Figure 8 These are the mechanical strength test results of the frozen microneedles and the thawed microneedles of the present invention.

[0040] Figure 9 These are the statistical results of the elastic modulus of the frozen microneedles and the thawed microneedles of the present invention.

[0041] Figure 10 This is a histological section of cells delivered via cryo-microneedle transdermal delivery according to the present invention.

[0042] Figure 11 This invention describes the distribution of live and dead cells in cryogenic microneedles and microneedles prepared by traditional drying methods.

[0043] Figure 12 This is a statistical result of the number of live cells and dead cells in the cryogenic microneedles of this invention and the microneedles prepared by the traditional drying method.

[0044] Figure 13 This is a three-dimensional reconstruction image of fluorescent traces of hair follicle organoid cells cultured in vitro in the cryo-microneedle of the present invention.

[0045] Figure 14 This is an experimental diagram illustrating the use of cryo-microneedles of the present invention to achieve breakthrough hair growth on the skin surface in animals.

[0046] Figure 15 It is a magnified view of the hair follicle image from a level perspective to reconstruct it.

[0047] Figure 16 It is a statistical chart of the proportion of reconstructed hair follicles.

[0048] Figure 17 The procedure involves reconstructing hair follicles, obtaining paraffin-embedded pathological sections, and staining with eosin-hematoxylin. Detailed Implementation

[0049] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0050] Unless otherwise specified, the experimental methods in the following examples are conventional methods; the chemical and biological reagents used are conventional reagents in the art and are commercially available unless otherwise specified.

[0051] Main reagent and material sources:

[0052] Type A pigskin gelatin powder: CAS No.: 9000-70-8.

[0053] Polydimethylsiloxane (PDMS) microneedle mold: Commercially available conventional PDMS microneedle mold is used.

[0054] Example 1

[0055] I. Preparation of methacrylated gelatin hydrogel precursor

[0056] Foam-like methacrylated gelatin hydrogel precursors can be prepared using commercially available hydrogel precursor products or by methods reported in existing technologies.

[0057] In this embodiment, methacrylated gelatin is prepared by mixing methacrylic anhydride with gelatin to form a methacrylated gelatin hydrogel solution. The preparation method is based on the literature (Kong B, Chen Y, Liu R, Liu X, Liu C, Shao Z, Xiong L, Liu X, Sun W, Mi S. Fiber reinforced GelMAhydrogel to induce theregeneration of corneal stroma. Nat Commun. 2020 Mar 18; 11(1):1435.), and the synthesis steps are as follows:

[0058] (1) Weigh 10g of type A porcine skin gelatin powder and 100mL of phosphate (PBS) buffer. Add 10g of type A porcine skin gelatin powder to 100mL of phosphate buffer and place it in a 50℃ water bath and stir continuously for 3 hours to fully dissolve it, thus preparing a 100ml / ml hydrogel precursor solution.

[0059] (2) Then, slowly add 6 mL of methacrylic anhydride to the hydrogel precursor solution at a rate of 0.1 mL / min and react at 50 °C for 3 hours.

[0060] (3) Subsequently, 500 mL of 40 °C phosphate buffer was added to dilute and terminate the methacrylation reaction.

[0061] (4) To further remove unreacted methacrylic anhydride and other components (impurities, harmful substances), the solution diluted with phosphate buffer was dialyzed at 40°C for 7 days using a dialysis bag (Solarbio, catalog number: YA1051) with a molecular weight cutoff of 12-14 kDa. The deionized water was changed every 12 hours.

[0062] (5) After the purified solution was placed in a -80℃ refrigerator overnight, it was freeze-dried in a vacuum freeze dryer at -105℃ for 2 days to obtain a white porous foam-like methacrylated gelatin hydrogel precursor without phosphate buffer and residual methacrylic anhydride. It was then stored in a dry sealed bag away from light.

[0063] II. Synthesis of cryohydrogels for carrying living cells

[0064] Follow these steps:

[0065] (1) Preparation of photocrosslinkable 100 mg / ml methacrylated gelatin hydrogel: Weigh 100 mg of the methacrylated gelatin precursor foam prepared in the previous step, and add 1 ml of phosphate buffer containing 1 mg / ml phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (CAS No.: 85073-19-4) photoinitiator.

[0066] (2) Place it in a 60℃ water bath and heat it in a water bath for 30 minutes, stirring once every 10 minutes to obtain methacrylated gelatin hydrogel that can be cross-linked under 405nm ultraviolet light, for use in step (3).

[0067] (3) Synthesis of cryohydrogel with cryopreservation solution: Take 1 ml of the methacrylated gelatin hydrogel obtained by heating and dissolving in step (2), add 1 ml of CryoStor CS10 cryopreservation solution (STEMCELL Technologies, catalog number: 07930, containing 100 mg / ml dimethyl sulfoxide) containing 100 ml / ml dimethyl sulfoxide in a centrifuge tube and mix thoroughly. Due to the proportional dilution, the final cryohydrogel with a final concentration of 50 mg / ml methacrylated gelatin and 50 mg / ml dimethyl sulfoxide is prepared.

[0068] (4) The frozen hydrogel obtained in step (3) is filtered through a 0.22 μm bacterial filter to form a sterile frozen hydrogel.

[0069] III. Analysis of Frozen Hydrogel Components and Groups using Fourier Transform Infrared Spectroscopy via Frozen Hydrogel Patch Method

[0070] Follow these steps:

[0071] (1) Take 1 mg of the dried synthetic cryogel and 1 mg of methacrylated gelatin hydrogel as test samples.

[0072] (2) Place each of the 200 mg of dry potassium bromide in a mortar and grind them thoroughly and evenly for at least 1 minute.

[0073] (3) Place the mixed sample into a tableting mold and press it under a pressure of 15MPa for 60s to obtain a semi-transparent tablet.

[0074] (4) Insert the mold sleeve and the insert plate of the sample ingot directly into the sample holder of the NicoLET iS 50FT-IR testing instrument for measurement.

[0075] The results are as follows Figure 1 The image shows the Fourier transform infrared (FTIR) spectra of a cryo-hydrogel (labeled Cryo-GelMA) and a methacrylated gelatin hydrogel (labeled GelMA). The vertical axis represents transmittance, and the horizontal axis represents wavenumber. (3317 cm⁻¹) -1 The spectrum at 3090 cm⁻¹ is a superposition of the OH stretching vibration and the NH stretching vibration. -1 The point is the CH2 stretching vibration; 2952 cm. -1 The vibration is CH3 antisymmetric stretching vibration; 1642 cm. -1 1553cm -1 1244cm -1 These are the typical characteristic absorption peak ranges of amides I-III in methacrylated gelatin; 1080 cm⁻¹ -1 and 1037cm -1 COH bending vibration; 695cm -1 This corresponds to the rocking vibration of water molecules. The general spectrum and amide groups of the cryogel are similar to those of methacrylated gelatin. Due to the introduction of cell cryopreservation solution, at 10¹⁸ cm⁻¹... -1 The strongest characteristic peak appears at this point, which corresponds to the antisymmetric stretching vibration of COC. Based on the overall spectrum analysis, it is an ether group.

[0076] IV. Extraction of primary skin-derived live dermal and epidermal cells

[0077] Follow these steps:

[0078] (1) Euthanasia of newborn mice (strain: C57BL / 6) and disinfection of their skin: Animals were euthanized using the drug inhalation method. Four newborn mice were placed in centrifuge tubes containing cotton balls soaked in ether in a fume hood. The tubes were sealed for 20 minutes. After euthanizing the mice, the ether was carefully discarded. The mice were then immersed in alcohol disinfectant and shaken for 2 minutes to remove impurities and bacteria from their skin. This disinfection was repeated twice. Finally, the mice were rinsed three times with phosphate buffer to remove excess alcohol.

[0079] (2) Peeling off intact newborn mouse skin: The skin of the newborn mouse was separated in a 60mm sterile culture dish using sterile dissecting tools. The limbs and tail of the newborn mouse were removed using ophthalmic scissors, leaving its intact trunk. The skin was cut along the midline of the mouse's back from the top of the head to the tail using ophthalmic scissors. Then, the skin was slightly separated from the subcutaneous tissue using ophthalmic forceps, the free skin was grasped and torn off from the tail to the head, completely peeling off the newborn mouse skin.

[0080] (3) Rinsing the separated skin: Add an appropriate amount of phosphate buffer to three sterile culture dishes. Place the separated skin with the dermal side down in the dishes and rinse to remove blood generated during the separation process and any remaining subcutaneous tissue or internal organs. Rinse thoroughly in the three culture dishes in turn until all skin has been collected and placed in a new culture dish.

[0081] (4) Neutral enzyme digestion and termination: Take 4 ml of 1 mg / ml neutral protease and preheat it in a 37°C water bath. Take 10 ml of the preheated 1 mg / ml neutral protease and add it to a culture dish containing the isolated newborn mouse skin. Spread the dish with the dermis side down so that the skin is completely immersed in the neutral protease. Incubate the dish at 37°C for 1 hour and add 4 ml of phosphate buffer to terminate the digestion.

[0082] (5) Separation of dermis and epidermis: Using ophthalmic forceps, press the dermis at the edge of a single skin sheet while holding the epidermis above it, and peel it off completely from the dermis. Place the white opaque epidermis and the brownish-red gel-like dermis in separate culture dishes.

[0083] (6) Collagenase consumption of dermis and termination: Use ophthalmic scissors to cut the dermal tissue into small pieces, add 4 ml of 2 mg / ml (w / v) type I collagenase to the dermal tissue, place it in an incubator at 37°C for 1 hour, and terminate digestion with 4 ml of phosphate buffer.

[0084] (7) Trypsin digestion of the epidermis and termination: Use ophthalmic scissors to cut the epidermal tissue into small pieces, add 4 ml of 2.5 mg / ml trypsin to the epidermal tissue, place it in an incubator at 37°C for 15 minutes, and terminate digestion with 4 ml of phosphate buffer.

[0085] (8) Large tissues were filtered using a cell filter with a diameter of 70 μm to obtain solutions containing live dermal stem cells and solutions containing live epidermal stem cells.

[0086] Example 2

[0087] I. Preparation of Layered Cryo-Microneedles for Transdermal Delivery of Living Cells

[0088] The sterile cryohydrogel prepared in step two of Example 1 and the solution containing living dermal and epidermal stem cells prepared in step four were used to prepare the cryoneedles of the present invention. The procedure was carried out according to the following steps (flowchart shown). Figure 2 As shown):

[0089] (1) Microneedle preparation: Polydimethylsiloxane (PDMS) microneedle molds were used uniformly for fabricating cryo-microneedles. Each microneedle mold, derived from the master mold, had a bottom diameter of 600-800 μm and a height of 1000-1200 μm. These needle cavities were arranged in a 5×5 array, with a needle tip-to-needle spacing of 2000 μm. All PDMS molds were soaked in 75% alcohol for 30 minutes and then placed in a biosafety cabinet for UV sterilization for 30 minutes to ensure aseptic standards were met.

[0090] (2) Cryo-needle perfusion: A solution containing dermal and epidermal stem cells was mixed in the same 15 mL centrifuge tube, with a cell ratio of epidermal stem cells to dermal stem cells of 1:2. The mixture was then centrifuged at 150 x g for 3 minutes. After discarding the supernatant, the cells were resuspended in 600 μL of liquid cryo-hydrogel, so that the total viable stem cell content in the cryo-hydrogel was 0.8–1.4 × 10⁻⁶ cells / mL. 6 Cells / mL. The cryogel containing live cells was injected into a polydimethylsiloxane microneedle mold. The microneedle mold containing the cell / hydrogel mixture was placed in a 6-well cell culture plate and centrifuged at 710xg for 3 minutes. This process removed the air trapped in the needle tip and concentrated the cells in the suspension at the tip of the mold needle.

[0091] (3) Filling the cryogenic substrate: Excess hydrogel and cells overflowing from the needle tip were aspirated using a pipette. The microneedle tip was irradiated with 405nm UV light to crosslink the materials for 12 seconds. For the microneedle substrate, fresh, sterile cell cryopreservation solution CryoStor CS2 (STEMCELL Technologies, catalog number: 07932, containing 20 mg / ml dimethyl sulfoxide) was added to the mold to fill the substrate. All the above perfusion molding operations and centrifugation processes were performed at 4°C to maintain cell viability.

[0092] (4) Programmed cooling step: Freeze the mold at -20℃ for 2 hours, gently peel the frozen microneedles off the mold on dry ice, place them in a 600mm sterile cell culture dish, then freeze at -80℃ for 2 hours, and then freeze in liquid nitrogen at -196℃ for 1 hour to obtain the frozen microneedles of the present invention. A schematic diagram of its structure is shown below. Figure 3 As shown in the image, the obtained cryo-microneedles are shown in the figure below. Figure 4As shown, methylene blue was added to the needle tip to distinguish the layered tip from the base for observation.

[0093] II. Cryo-microneedle scanning electron microscopy detection of the present invention

[0094] (1) Pretreatment before scanning electron microscopy: The prepared cryogenic microneedles were freeze-dried in a vacuum freeze dryer at -80℃ for 24 hours.

[0095] (2) The freeze-dried microneedle sample was transferred to a vacuum for sputtering to coat the sample surface with conductive metal.

[0096] (3) Use double-sided carbon conductive tape to attach the microneedle base, adjust the preset shooting droplet, stick it on the sample stage to increase conductivity, place it in the scanning electron microscope for observation, and take pictures to record.

[0097] III. Scanning electron microscopy detection of traditional microneedles prepared by heat drying

[0098] To compare conventional microneedles prepared by heat drying with the cryogenic microneedles of this invention, conventional microneedles were prepared according to the following method and examined by scanning electron microscopy:

[0099] (1) Add 10% (w / v) methacrylated gelatin to the PDMS microneedle mold and place it in a vacuum environment at 50°C to remove bubbles.

[0100] (2) After centrifuging at 1000xg for 5 minutes, the microneedle mold carrying hydrogel was exposed to ultraviolet light for cross-linking for 30 seconds.

[0101] (3) Place the mold in an oven at 30-35℃ and dry for 24 hours to separate the microneedle array from the microneedle mold.

[0102] (4) The heat-dried microneedle device is moved to a vacuum for spray coating so that the sample surface is coated with conductive metal.

[0103] (5) Use double-sided carbon conductive tape to attach the microneedle base, adjust the preset shooting droplet, stick it on the sample stage to increase conductivity, place it in the scanning electron microscope for observation, and take pictures to record.

[0104] Scanning electron microscope images of the cryogenic microneedles and conventionally prepared microneedles prepared by heat drying according to the present invention are shown below. Figure 5 As shown: The cryogenic microneedles for carrying cells of the present invention exhibit a dense and porous surface microstructure, which facilitates material interaction and intercellular communication among living cells, promotes cell migration and aggregation, and facilitates metabolism, providing a better carrier medium for living cells. In contrast, microneedles prepared by traditional drying methods have a smooth, dense, and non-porous surface, lacking the space for cell migration and aggregation as well as the pores for material exchange.

[0105] Example 3: Performance testing of the cryo-microneedles of the present invention

[0106] The performance of the cryo-microneedles of the present invention prepared in Example 2 was tested:

[0107] I. Evaluation of Morphological Maintenance After Thawing of Cryo-Microneedles

[0108] (1) Place the cryo-microneedle with the tip facing up in an open environment at room temperature.

[0109] (2) At 5 seconds, 30 seconds, 60 seconds, 120 seconds and 180 seconds, a high-definition camera was used to observe and photograph the surface of the microneedle device.

[0110] like Figure 6 As shown, after cryogenic freezing, microneedles come into contact with water molecules in the air, forming white ice crystals on the tip surface. After 180 seconds, the cryogenically frozen microneedles prepared by cryogel completely melted, but still maintained a good shape. This indicates that hydrogels that have lost the strength of ice crystals still have a certain strength to support the maintenance of their shape under the hydrogen bonds provided by photocrosslinking.

[0111] II. Cryo-microneedle mechanical strength testing

[0112] (1) To evaluate the mechanical force of the cryo-microneedles of the present invention, the cryo-microneedles of the present invention and the cryo-microneedles after melting (hereinafter referred to as the melted microneedles) are compared.

[0113] (2) The thawed cryogel microneedles were obtained by a complete programmed cooling process: freezing at -20°C for 2 hours, freezing at -80°C for 2 hours, and freezing in liquid nitrogen at -196°C for 1 hour. After being removed from the liquid nitrogen, they were placed at room temperature for at least 3 minutes until the cryogel material exhibited a uniform and transparent gel microneedle morphology.

[0114] (3) Pre-cooling the material mechanics measurement system: Before testing, place a flat-bottomed stainless steel disc on the platform of the material mechanics measurement system (Ametek LS1, USA) and pre-cool the stainless steel disc with liquid nitrogen at -196°C for at least 3 minutes, replenishing the liquid nitrogen as needed.

[0115] (4) Place the cryo-microneedle flat with the tip facing upwards, and use a flat-headed stainless steel cylindrical sensor with a diameter of 5 mm to apply the force vertically downwards at a constant speed of 0.5 mm per minute. The force is applied vertically downwards to the tip of the microneedle.

[0116] The compressed shape of cryo-microneedles is as follows Figure 7 As shown: the needle tip is compressed and flattened, undergoing deformation.

[0117] The mechanical strength test results of the microneedles are as follows Figure 8As shown: The cryo-microneedles of the present invention have no obvious break points, indicating that the cryo-microneedles with ice crystal structure have good resistance to deformation in the needle body.

[0118] The elastic modulus of the microneedles was calculated, and the results are as follows: Figure 9 As shown, compared with the melted microneedles, the ice crystals formed by cryogenic freezing increased the stiffness of the microneedles by 110 times, indicating that freezing endows the microneedles with excellent mechanical properties.

[0119] III. Transdermal Ability Testing of Cryomicroneedles

[0120] (1) Pre-labeling cells: First, before loading the cells into the microneedle mold, add 5 μL of NucBlue LiveReadyProbes (Invitrogen, USA) nuclear dye to the cell suspension and incubate at 37°C in the dark for 15 minutes. After incubation, centrifuge at 170 x g for 3 minutes and wash three times with phosphate buffer.

[0121] (2) After the final centrifugation and discarding of the supernatant, add cryogel into the centrifuge tube to resuspend the cells, then inject into the mold, and add base. CS2 cryopreservation solution.

[0122] (3) After programmed cooling, the prepared frozen microneedles with NucBlue labeled cells were taken out of liquid nitrogen and quickly inserted into the skin of pigs and nude mice by pressing with the thumb.

[0123] (4) After the microneedle base melts, use tissue embedding agent to vertically embed the skin tissue.

[0124] (5) After freezing in a -20℃ freezer, the frozen sections were observed under a fluorescence microscope (Zeiss, AxioImager D2, Germany).

[0125] Cryo-microneedles, labeled with nuclear dyes to represent stem cells, were applied to mouse skin for transdermal delivery. Pathological examination and microscopic observation revealed... Figure 10 As shown, the ability of cryo-microneedles to deliver cells transdermally has been confirmed.

[0126] IV. Cell viability detection in cryo-microneedles

[0127] (1) After taking the cryogel-prepared cryoneedles out of liquid nitrogen using tweezers, place them in a confocal dish, float the confocal dish in a 37°C water bath, and heat them in water to revive them.

[0128] (2) Perform live / dead cell staining detection according to the Calcein-AM / PI live / dead cell staining kit (brand: Solarbio, catalog number: CA1630). In short, prepare the staining working solution according to the Calcein-AM / PI live / dead cell staining kit. Add 1 μL of Calcein-AM dye and 1 μL of PI dye to 1 mL of the reagent-matched staining buffer solution, and adjust the total amount of working solution accordingly.

[0129] (3) After incubating the microneedles in the confocal dish at 37°C in a dark oven for 30 minutes, observe and photograph them under an LSM980 laser confocal microscope and perform three-dimensional reconstruction.

[0130] (4) Image statistical analysis: Cell viability was calculated by ImageJ image analysis and histogram plotted by GraphPad Prism.

[0131] To further demonstrate that the microneedles prepared by the conventional drying method in Example 2 cannot be used for stem cell delivery, and that the frozen microneedles of the present invention have the ability to maintain living cells, the viability of cells in the microneedles prepared by the two methods was evaluated by cell viability staining. The results are as follows: Figure 11 , 12 As shown, the microneedles prepared by the cryogel method still retain more than 70% viable cells; while the microneedles prepared by the heat drying method lose all cell viability and contain no viable cells. Experiments confirm that the cryo-microneedles of this invention can be used to deliver substances such as live cells.

[0132] V. Live Cell Delivery and Fluorescent Tracing via Cryomicroneedles

[0133] (1) Confocal dish molding: In order to observe the migration and differentiation of DCs and ECs in the microneedles after cryopreservation and thawing, 10:1 PDMS and matching curing agent were pre-filled into the confocal dish, and a PMDS mold with the same microneedle shape was constructed in the confocal culture dish using a conical microneedle array master mold.

[0134] (2) Cell fluorescent labeling: 5 μL of green fluorescent tracer CellTracker was added. TM CM-DiO was added to a dermal cell suspension, along with 5 μL of the red fluorescent tracer CellTracker. TM CM-DiI was added to the epidermal cell suspension and incubated at 37°C in the dark for 20 minutes, followed by rinsing three times with phosphate buffer.

[0135] (3) Preparation of layered cryo-microneedle device: After cell staining, the layered cryo-microneedle device was prepared according to the preparation method in Example 4.

[0136] (4) Delivery to the confocal dish mold: Insert the layered microneedles, which have been frozen in liquid nitrogen, into the confocal mold.

[0137] (5) Add culture medium: After the substrate is completely dissolved, add 1 ml of 10% FBS / DMEM (v / v) culture medium, let stand for 30 minutes and then change the medium. Then change the medium every 2 days.

[0138] (6) Confocal microscopy and three-dimensional image reconstruction: The changes in culture were observed and photographed under a laser confocal microscope (Zeiss, LSM980, Germany), and the images were reconstructed in three dimensions.

[0139] Dermal cells were labeled with a green fluorescent probe and epidermal cells were labeled with a red fluorescent probe. Figure 13 In Figure A, it can be clearly observed that initially, most cells are densely distributed at the bottom, but some scattered cells remain at a distance of 600 μm from the needle tip. Subsequently, the scattered cells gradually aggregate, forming aggregates with a diameter of approximately 100 μm. The results indicate that cells thawed and cultured in the three-dimensional environment provided by the cryogel possess the ability to further aggregate and self-assemble, effectively protecting and maintaining cell-cell interactions, cell-specific self-assembly capabilities, and differentiation potential, thus providing a suitable three-dimensional environment for the cells.

[0140] Further observation under an inverted microscope, such as Figure 13 B. It can be seen that after the cells are delivered to the confocal microneedle model, the cells are initially discrete on day 0, then form cell clumps on day 3 and day 7, and a dark brown bud-like structure is visible on day 14. This confirms that the cells delivered by the cryo-gel-prepared cryo-microneedles have the ability to further self-assemble and differentiate.

[0141] Example 4: Hair follicle reconstruction using the cryo-microneedles of the present invention

[0142] (1) Preservation of cryogenic microneedles: The cryogenic microneedles of the present invention prepared in Example 2 were temporarily preserved in a stainless steel cotton liquid nitrogen basin containing liquid nitrogen at -196°C.

[0143] (2) Anesthesia and disinfection of animals: Nu mice (strain: Nu / NuNude, age: 7 weeks) were first anesthetized by intraperitoneal injection of 10 mg / ml pentobarbital. After the nude mice were completely anesthetized, the skin of the mice was disinfected by wiping the middle part of the back of the nude mice with 75% alcohol swabs.

[0144] (3) Pressing the cryo-microneedle for transdermal delivery of live cells: Use a stainless steel, cotton-lined liquid nitrogen basin to move the cryo-microneedle next to the nude mouse, minimizing the contact time of the microneedle at room temperature. Use your left hand to spread the skin on the back of the nude mouse, flattening the skin to achieve a certain degree of tension. Use tweezers to remove the cryo-microneedle from the liquid nitrogen and place it on the mouse skin surface with the needle tip facing down. Use your thumb to quickly and evenly press the base of the microneedle until you feel a slight breakthrough.

[0145] (4) Press the microneedle base continuously for about 3-5 minutes. After the base melts, wipe off the excess liquid to complete the transdermal delivery of loaded live cells.

[0146] In this embodiment, to evaluate the maintenance of stem cell differentiation potential using cryo-microneedles, a xenograft mouse model was used. The prepared cryo-microneedles carrying hair follicle organoids were pressed onto the back skin of nude mice, delivering the organoids into the dermis. After 14 days, a matrix-distributed pattern of black hair shafts was visible breaking through the skin surface of the nude mice and growing. Figure 14 As shown, the cryo-microneedle delivery of live cells of the present invention has been confirmed, which can achieve further differentiation of stem cells and enable minimally invasive hair follicle reconstruction in the field of hair follicle reconstruction.

[0147] Further magnification and reconstruction of hair follicles from a level perspective, such as... Figure 15 As shown, the reconstructed sites darkened in color on day 7, and clusters of hair broke through the skin surface to grow on day 14. The proportion of reconstructed hair follicles is as follows. Figure 16 As shown, out of 25 sites, 11 sites showed hair growth breaking through the skin surface. After paraffin-embedded pathological sections and eosin-hematoxylin staining of the reconstructed hair follicles, as shown... Figure 17 As shown, in the center of the slice image, the reconstructed black hair breaks through the stratum corneum of the epidermis and grows. The reconstructed hair follicle structure is different from that of the nude mouse itself.

Claims

1. A method for preparing cryo-microneedles for transdermal delivery of living stem cells, characterized in that, Includes the following steps: I. Preparation of foam-like methacrylated gelatin hydrogel precursor; II. Synthesis of cryo-hydrogels: Take the foam-like methacrylated gelatin hydrogel precursor prepared in step one, add buffer A containing a photoinitiator, heat to dissolve, and obtain a methacrylated gelatin hydrogel with a concentration of 80~120 mg / ml; take the methacrylated gelatin hydrogel, add CryoStor CS10 cell cryopreservation solution, mix well, sterilize, and obtain a sterile cryogel; the volume ratio of methacrylated gelatin hydrogel to CryoStor CS10 is 1:0.8~1.2; III. Preparation of cryo-microneedles, including the following steps: (1) Cryo-needle tip perfusion: Centrifuge the prepared solution containing live stem cells and discard the supernatant. Resuspend the live stem cells in the prepared cryo-hydrogel. Inject the cryo-hydrogel containing live stem cells into the needle cavity of the sterile microneedle mold. Then centrifuge the microneedle mold to remove the air trapped in the needle tip and make the live stem cells in the suspension densely concentrated at the tip of the mold needle. (2) Filling the cryogenic substrate: Perform the following operations at 4~8℃: remove excess hydrogel from the microneedle mold, irradiate the microneedle tip with ultraviolet light to crosslink the raw materials, then add the cell cryopreservation solution CryoStor CS2 to the microneedle mold to fill the substrate, and then centrifuge. (3) Programmed cooling and freezing: The mold is subjected to programmed cooling and freezing, and then demolded to obtain frozen microneedles. The specific method is as follows: the mold is frozen at -20℃ ~ -30℃ for 2~4 hours, the frozen microneedles are demolded from the mold at low temperature, then frozen at -70℃ ~ -90℃ for 2~4 hours, and finally frozen in liquid nitrogen for 1~3 hours to obtain frozen microneedles.

2. The preparation method according to claim 1, characterized in that: In step one, a foam-like methacrylated gelatin hydrogel precursor is prepared using methacrylic anhydride and gelatin as raw materials. The gelatin is selected from pig skin gelatin powder, fish skin gelatin powder, fish scale gelatin powder, cow skin gelatin powder, cow bone gelatin powder, and chicken skin gelatin powder; the ratio of methacrylic anhydride to gelatin is 5~7 mL : 10g.

3. The preparation method according to claim 1, characterized in that: The photoinitiator in step two is selected from lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, 2,2-dimethoxy-2-phenylphenylphenylethyl, Irgacure-2959, Irgacure-184, and Irgacure-907, and the buffer solution A is a phosphate buffer, a sodium bicarbonate buffer, or a sodium citrate buffer. The concentration of the methacrylated gelatin hydrogel is 90~110 mg / ml, of which the concentration of the photoinitiator is 0.8~1.2 mg / ml; the volume ratio of the methacrylated gelatin hydrogel to CryoStor CS10 is 1:0.9~1.

1.

4. The preparation method according to claim 3, characterized in that: In step three In step (2) of freezing the substrate filling, the raw material is cross-linked by irradiating the tip of the microneedle with ultraviolet light for 12 to 60 seconds. The specific steps of step (3) of programmed cooling are as follows: the mold is frozen at -20°C for 2 hours, the frozen microneedles are gently peeled off the mold on dry ice and placed in a sterile container, then frozen at -80°C for 2 hours, and then frozen in liquid nitrogen at -196°C for 1 hour to obtain the frozen microneedles.

5. The preparation method according to claim 1, characterized in that: The bottom diameter of each needle in the microneedle mold is 600-800μm, the height is 1000-1200μm, and the distance between needle tips is 1500-2500 μm.

6. The preparation method according to claim 1, characterized in that: The living stem cells include hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, liver stem cells, muscle satellite cells, skin stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, and embryonic stem cells; the cell content in the cryogel containing living stem cells is 0.8~1.4×10⁻⁶. 6 mL -1 .

7. A cryo-microneedle for transdermal delivery of living stem cells, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 6.

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