A stem cell preparation sponge patch complex and its preparation method

Abstract

The present invention discloses a stem cell preparation sponge patch complex and a preparation method thereof. The sponge patch complex is a degradable sponge body infiltrated with a stem cell suspension, and the degradable sponge body is made of gelatin, modified sodium alginate, and polyethylene glycol-polylysine-polyvaline triblock copolymer; the mass ratio of the gelatin, modified sodium alginate, and polyethylene glycol-polylysine-polyvaline triblock copolymer is 5:(1-6):(1-6). In the present invention, a degradable sponge body is prepared with modified sodium alginate and polyethylene glycol-polylysine-polyvaline triblock copolymer, which can accommodate more stem cells, improve the stem cell loading capacity, and L-carnitine and vitamin E are added to the complete medium for neural stem cells, thereby improving the survival rate of stem cells.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedical and tissue engineering research, and particularly relates to a stem cell preparation sponge patch complex and a preparation method thereof. Background Art

[0002] Brain diseases refer to nerve tissue damage caused by genetics, congenital brain hypoplasia, brain trauma, brain tumors, cerebral hemorrhage, cerebral infarction, chemical drug poisoning, etc., covering organic brain damage with abnormal tissue structure and physiological functions. According to the disease causes, brain diseases are divided into three categories: one is cerebral palsy, mental retardation, etc. caused by genetics and congenital dysplasia. One is sequelae of acute brain injury caused by trauma, sequelae of stroke caused by cerebrovascular diseases, etc. Another is chronic degenerative diseases caused by central nerve damage and subsequent senescence and degeneration of nerve cells, including Alzheimer's disease, brain atrophy, Parkinson's disease, etc. In China, there are about 10 million new cases of brain diseases every year, and the lethality and disability rate is about 75%. The medical expenses for the treatment of encephalopathy by the country and patient families reach tens of billions of yuan, and it has become one of the major diseases endangering national health.

[0003] Ultimately, brain diseases are nerve tissue damage. As the basic structural and functional units of the nervous system, nerve cells, due to their high degree of differentiation, once damaged, require an extremely slow repair process in mild cases, and irreversible damage in severe cases. Facing the worldwide problem of nerve tissue injury repair, stem cell therapy provides a new approach. Stem cells implanted into the cranial cavity through the intranasal route have become an emerging transplantation method and have received increasing attention. The advantages of stem cell transplantation through the intranasal route include: non-invasive; there is a unique direct channel between the nasal cavity and the brain anatomically. After nasal administration, the drug can bypass the blood-brain barrier and enter the central nervous system, that is, cerebrospinal fluid or brain tissue, to play a therapeutic role after being absorbed through the nose; the nasal mucosa has rich blood vessels and lymphatic vessels, with a large absorption area and convenient conditions for entering the cranial cavity.

[0004] The patent 201810623850.5 previously applied for by the inventor disclosed a stem cell preparation sponge patch complex for treating brain diseases, a preparation method thereof, and an application. This patent application used gelatin as the raw material for preparing the sponge patch. Although the prepared stem cell preparation sponge patch complex has a high porosity, the rigidity between the voids is weak, resulting in the rupture of stem cells during the process of squeezing the material into the lesion, ultimately leading to a decrease in the survival rate of stem cells, and the number of stem cells carried in the pores is small, resulting in a low stem cell loading. Summary of the Invention

[0005] The purpose of the present invention is to provide a stem cell preparation sponge patch complex and a preparation method thereof.

[0006] A stem cell preparation sponge patch complex, wherein the sponge patch complex is a degradable sponge body infiltrated with a stem cell suspension, and the degradable sponge body is made of gelatin, modified sodium alginate, and a polyethylene glycol-polylysine-polyvaline triblock copolymer; the mass ratio of the gelatin, modified sodium alginate, and polyethylene glycol-polylysine-polyvaline triblock copolymer is 5:(1-6):(1-6).

[0007] The stem cells are neural stem cells.

[0008] The modified sodium alginate is prepared by the following method: dissolving sodium alginate in an aqueous solution with a mass 15-30 times that of sodium alginate, adjusting the pH to 3-5, adding N-hydroxysuccinimide, stirring at 20-30 °C for 1-3 h, then adding L-cysteine methyl ester hydrochloride, raising the temperature to 50-70 °C, continuing to stir and react for 1-3 h, stopping stirring, performing sedimentation with ethanol, filtering to collect the precipitate, washing the precipitate with ethanol 2-4 times, and drying in vacuum for 3-5 h to obtain the modified sodium alginate.

[0009] The molar ratio of the carboxylate group in the sodium alginate, N-hydroxysuccinimide, and L-cysteine methyl ester hydrochloride is 3:(0.5-3):(0.5-3).

[0010] The density of the stem cell suspension is 8×10 6 -4×10 7 cells / mL.

[0011] The porosity of the degradable sponge body is more than 85%.

[0012] The preparation method of the stem cell preparation sponge patch complex includes the following steps: pre-soaking the degradable sponge body with a culture medium, then taking the pre-prepared single cell suspension of neural stem cells, diluting the suspension to a cell density of 8×10 6 -4×10 7 cells / mL, and fully infiltrating the degradable sponge body with it until it is saturated with absorption.

[0013] The components of the complete medium for neural stem cells are: 1% B27, 1% glutamine, 1% penicillin-streptomycin, 20 μg / ml bFGF, 20 μg / ml EGF, 50 IU / ml heparin, and the balance is water.

[0014] The culture medium is the complete medium for neural stem cells added with 1 μg / mL of L-carnitine and 3 μg / mL of vitamin E.

[0015] Advantages of the present invention: The stem cell preparation sponge patch complex of the present invention, due to having a certain rigid support, can be directly delivered to the optimal physiological site for cell therapy under the clamping of an instrument, and this process is non-invasive, effectively reducing the pain of patients. The stem cell preparation sponge patch complex of the present invention has the ability to deform while having rigidity. After being delivered to the treatment site, it can be positioned and stuck at this site, thus effectively solving the problem that in the current intranasal transplantation route, due to the mucociliary clearance of the nasal mucosa, the liquid medicines of existing nasal dosage forms, including nasal drops and gels, are unevenly distributed in the nasal cavity and are easily lost from the nasal cavity; the in-situ release of this cell preparation can better promote the absorption of the stem cell preparation and the exertion of the drug effect. The present invention prepares a degradable sponge body with modified sodium alginate and polyethylene glycol-polylysine-polyvaline triblock copolymer, which can accommodate more stem cells, increase the stem cell loading, and add L-carnitine and vitamin E to the complete medium for neural stem cells, improving the survival rate of stem cells. Description of the Drawings

[0016] Figure 1 Cultivation morphology diagrams of neural stem cells of the same batch and different passages.

[0017] Figure 2 Flow cytometer detection result diagrams of neural stem cell characteristic proteins Vimentin, Sox2, Nestin, SSEA1, Notch1, and Musashi1.

[0018] Figure 3 Growth curve diagrams of neural stem cells of different passages.

[0019] Figure 4 Immunofluorescence detection result diagrams of neural stem cell characteristic proteins Nestin, Vimentin, Sox1, Sox2, Musashi, Notch1, CXCR4, and SSEA1. Detailed Embodiments

[0020] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0021] Example 1

[0022] Collection and transportation of the tissue source of neural stem cells:

[0023] 1) The tissue source is aborted fetuses (medical waste) at 8 - 14 gestational weeks, and an informed consent form for "Collection and Preservation of Donor Tissue Samples" is signed.

[0024] 2) The relevant medical staff select a container according to the tissue size, place it in a sterile tube, seal the tube mouth with a sealing film, wipe the outer surface of the tube with alcohol, and then put it into a transport box.

[0025] 3) During transportation, the vehicle should be kept stable to avoid the box being impacted. The inside of the transport vehicle should be clean, and the temperature inside the vehicle should be controlled below 20°C, and the temperature in the transport box should be about 4°C.

[0026] Primary preparation of neural stem cells:

[0027] 1) Place the intact fetus in a 100-mm sterile Petri dish and rinse it repeatedly with PBS buffer.

[0028] 2) Peel off the head skin and bones, open the cranial cavity to expose the brain tissue, and use fine surgical forceps to tear off the vascular membrane around the brain tissue.

[0029] 3) Clamp the edge of the cerebral cortex, separate the cerebral cortex, and place it in the neural stem cell complete culture medium solution in a 35-mm sterile Petri dish. Divide the cerebral cortex into tissue blocks about 1 mm in size.

[0030] 4) Transfer the obtained fetal cerebral cortex and neural stem cell complete culture medium solution into a 1-mL centrifuge tube with a dropper, slowly pipette and blow 30 times with a pipettor, let it stand for 10 minutes to make the undispersed tissue blocks precipitate to the bottom of the tube, and then aspirate the supernatant.

[0031] 5) Centrifuge at 820 rpm / min, aspirate and discard the supernatant, add 2 ml of sterile digestive solution, pipette and blow evenly, and place it in a 35-mm culture dish.

[0032] 6) Place the culture dish in a 37°C, 5% CO2 incubator for digestion for 1 - 2 minutes, add an equal volume of neural stem cell complete culture medium solution to terminate digestion, and centrifuge at 820 rpm / min and 20°C for 5 minutes.

[0033] 7) Use a dropper to aspirate the supernatant to above the cell precipitate line, add 5 mL of neural stem cell complete culture medium solution with a pipette, pipette up and down 3 times to resuspend the cells, and centrifuge at 820 rpm / min and 20°C for 5 minutes.

[0034] 8) Use a dropper to aspirate and discard the supernatant to above the cell precipitate line, use a 1-mL pipette tip to aspirate 1000 μL of neural stem cell complete culture medium solution and add it to the centrifuge tube, gently pipette and blow 10 - 15 times to form a single-cell suspension and perform cell counting.

[0035] 9) Add the calculated neural stem cell culture medium to the cells, pipette and blow into a single-cell suspension, with an inoculation density of 2 - 5×10 5 / mL, add it to a cell culture flask, mark the cell batch number, passage number, and cell quantity in the upper left corner of the culture flask, and mark the operator's name and operation date in the lower right corner.

[0036] 10) After inoculation, observe under an inverted microscope to ensure that the cells are evenly distributed in each culture container. If the cells are not evenly distributed, shake the container again and record the cell status.

[0037] 11) The inoculated and microscopically examined cell culture container is quickly placed in a carbon dioxide incubator at a temperature of 37°C and a CO2 concentration of 5%.

[0038] Neural stem cell passaging:

[0039] 1) Transfer the neurosphere suspension to be passaged into a 50 mL centrifuge tube using a pipette, rinse the culture container with an appropriate amount of PBS buffer, transfer the rinse solution into a 50 mL centrifuge tube, and let it stand for 10 minutes; collect the precipitated cell spheres.

[0040] 2) Add 1 ml of digestion solution to the cells, place the centrifuge tube in a 37°C, 5% CO2 incubator, digest for 2 min, add 4 mL of neural stem cell complete culture medium solution, neutralize, centrifuge at 820 rpm / min, 20°C for 5 min, aspirate the supernatant into a sterile container, mark it, and store it in a -80 degree refrigerator (conditioned medium).

[0041] 3) Use a pipette to remove the supernatant to about 200 μL of the scale line, flick the cell pellet, add 5 mL of neural stem cell complete culture medium solution, and centrifuge at 820 rpm / min and 20°C for 5 min.

[0042] 4) Re-seed at an appropriate density and continue culturing the cells in a 37°C, 5% CO2 incubator.

[0043] Release testing:

[0044] 1) Collect 25 mL of stem cell preparation cell suspension and aseptically inject it into aerobic blood culture bottles, anaerobic blood culture bottles and fungal blood culture bottles. After thorough mixing, seal with sealing film, mark the cell batch, operator name, operation time, and send for inspection immediately.

[0045] 2) Take three sterile tubes for testing, inject 3 ml of cell culture medium into each tube using a syringe in a sterile manner, seal with sealing film, mark the cell batch, operator name, operation time, and send to the laboratory. It is used for the detection of mycoplasma, chlamydia and related pathogenic microorganisms.

[0046] 3) Aseptically take 3 ml of culture medium and add it to a special tube for endotoxin detection. Seal the tube with sealing film, mark the cell batch, operator name, operation time, and send it for inspection immediately.

[0047] 4) Products can only be put into storage and released if the test reports show negative.

[0048] Neural stem cell packaging:

[0049] 1) Cell collection: Pipette all samples in the culture flask into a 50 ml centrifuge tube and centrifuge at 820 rpm / min and 20°C for 5 min.

[0050] 2) Preparation of single-cell suspension: Use a pipette to remove the supernatant to about 200 μL of the scale line, add 2 ml of neural stem cell complete culture medium solution, and use a 1 ml pipette tip to repeatedly pipette the cell pellet 30 times to physically disperse it into a single-cell suspension, and centrifuge at 820 rpm / min and 20°C for 5 minutes.

[0051] 3) Cell counting: Aspirate and discard the supernatant, add 1 mL of PBS buffer, gently tap the bottom of the tube to disperse and mix the cells, gently pipette 4 times to mix, and take 10 μL of single-cell suspension for counting.

[0052] 4) Adjustment of cell density: According to the counting results, add an appropriate amount of PBS buffer to adjust the concentration of the cell suspension to the target concentration.

[0053] 5) Cell sieving: Prepare another sterile 50mL centrifuge tube, take out the cell sieve, hold the edge with tweezers and place it at the mouth of the centrifuge tube, draw the cell suspension with adjusted density through the cell sieve, and collect it into the prepared centrifuge tube.

[0054] 6) Cell packaging and sampling: Take the prepared 2 mL cryotube, add the cell suspension, screw on the tube cap, seal with sealing film, and take 0.5 mL of cell suspension into 1 2 mL cryotube, mark it, seal with sealing film, and freeze at -80℃ for sample preparation.

[0055] 7) Preparation of cell preparation labeling: Label the prepared cell preparation and store it in a 4°C refrigerator until use.

[0056] Neural stem cell identification:

[0057] 1) Cell morphology detection: Single neural stem cells are spherical. When inoculated with single cells, under culture conditions, as the cells grow, they will spontaneously aggregate and grow into uniform spherical clones. Figure 1 shown.

[0058] 2) Flow cytometric detection of cell markers: Take 1×10 cells 6 The results of flow cytometry detection of cells expressing neural stem cell characteristic proteins Vimentin, Sox2, Nestin, SSEA1, Notch1, and Musashi1 are as follows Figure 2 As shown, the Nestin+ / Sox2+ cell ratio was greater than 90%.

[0059] 3) Cell proliferation ability: Add 100 μL of neural stem cell suspensions of different culture batches and different passages into a 96-well plate, with a cell concentration of 1000 cells / well. Incubate the culture plate in an incubator for 2 - 14 days. Add 10 μL of CCK-8 solution to each well, incubate the culture plate in the incubator for 2 h, and detect the absorbance value at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Use the data to create a curve with the culture time as the abscissa (X-axis) and the OD value as the ordinate (Y-axis). The doubling time of the cells can be calculated based on this curve to reflect the cell proliferation ability ( Figure 3 ).

[0060] 4) Immunofluorescence detection of cell markers: Take the human neural stem cell suspension, collect the cells, and rinse them 3 times with PBS and fix them with paraformaldehyde. Stain with neural stem cell-specific markers Nestin, Vimentin, Sox1, Sox2, Musashi, Notch1, CXCR4, and SSEA1, as Figure 4 shown.

[0061] Cell seeding:

[0062] 1) Add the corresponding volume of neural stem cell medium according to the type of cell culture container used. The neural stem cell medium is divided into 4 groups. One group is the ordinary neural stem cell medium, one group is the ordinary neural stem cell medium added with L-carnitine (final concentration 4 μg / mL), one group is the ordinary neural stem cell medium added with vitamin E (final concentration 4 μg / mL), and one group is the ordinary neural stem cell medium added with L-carnitine (final concentration 1 μg / mL) and vitamin E (final concentration 3 μg / mL); add 2 mL to each well in a low-attachment 6-well plate, add 5 mL / bottle to a T25 cell culture flask, add 15 mL / bottle to a T75 cell culture flask, and add 50 ml / bottle to a T175 cell culture flask.

[0063] 2) Add the counted cell suspension into the culture container, rinse the container of the original cell suspension with an appropriate amount of medium, transfer it together, and add a certain volume of cell medium. Manually shake the cell suspension evenly. Use a pipette with an appropriate measuring range to pipette under the liquid surface again, mark the cell batch number and cell quantity in the upper left corner of the culture flask, and mark the operator's name and operation date in the lower right corner.

[0064] 3) Observe under an inverted microscope to ensure that the cells are roughly evenly distributed in each culture container. If the cell distribution is uneven, shake the container again. Record the cell status and the medium condition by imaging every two days. This microscopy process should be completed within 5 minutes.

[0065] 4) Immediately place the cell culture container after inoculation and microscopy into an incubator at 37°C with 5% CO2. Pay attention to keeping the container level during movement. If it is a well plate, keep the well plate covered at all times during movement. If the container is a culture flask, when placing it in the carbon dioxide incubator, the bottle mouth should face left or right, rather than outwards. The container should not be placed close to the door, but should be placed slightly inside to reduce the risk of contamination when opening and closing the door.

[0066] Example 2

[0067] Preparation of modified sodium alginate: Dissolve sodium alginate in an aqueous solution with a mass 20 times that of sodium alginate, adjust the pH to 4.5, add N-hydroxysuccinimide, stir at 25°C for 2 h, then add L-cysteine methyl ester hydrochloride, raise the temperature to 60°C, continue stirring and reacting for 2 h, stop stirring, carry out sedimentation with ethanol, filter under suction to collect the precipitate, wash the precipitate 3 times with ethanol, and vacuum dry for 4 h to obtain modified sodium alginate. The molar ratio of carboxylate groups in the sodium alginate to N-hydroxysuccinimide and L-cysteine methyl ester hydrochloride is 3:1:1.

[0068] The preparation method of the polyethylene glycol-polylysine-polyvaline triblock copolymer refers to the method disclosed in Patent 201610345388.8, and the gelatin used is commercially available medical gelatin.

[0069] The mass ratio of the amounts of gelatin, modified sodium alginate, and polyethylene glycol-polylysine-polyvaline triblock copolymer is 5:3:3. Before use, wash the raw materials 3 times with distilled water, stir and mix the above three raw materials evenly to prepare a mixed material, add water with a weight 5 times that of the mixed material, stir evenly, inject into a mold and vacuum package, and crosslink with electron beam radiation at 30 kGy to obtain a hydrogel; soak the hydrogel in sterile water for 36 h until swelling equilibrium is reached to obtain a water-containing porous material, and freeze-dry the water-containing porous material at -40°C to obtain a degradable sponge.

[0070] Example 3

[0071] Preparation of modified sodium alginate: Dissolve sodium alginate in an aqueous solution with a mass 20 times that of sodium alginate, adjust the pH to 4.5, add N-hydroxysuccinimide, stir at 25°C for 2 h, then add L-cysteine methyl ester hydrochloride, raise the temperature to 60°C, continue stirring and reacting for 2 h, stop stirring, carry out sedimentation with ethanol, filter under suction to collect the precipitate, wash the precipitate 3 times with ethanol, and vacuum dry for 4 h to obtain modified sodium alginate. The molar ratio of carboxylate groups in the sodium alginate to N-hydroxysuccinimide and L-cysteine methyl ester hydrochloride is 3:1:1; the gelatin used is commercially available medical gelatin.

[0072] The mass ratio of the usage amounts of gelatin and modified sodium alginate is 5:6. Before use, the raw materials are washed 3 times with distilled water. The above two raw material substances are stirred and mixed evenly to prepare a mixed material. Water with a weight portion 5 times that of the mass of the mixed material is added, stirred evenly, injected into a mold and then vacuum-sealed, and crosslinked by electron beam radiation at 30 kGy to obtain a hydrogel; the hydrogel is soaked in sterile water for 36 h until swelling equilibrium is reached to obtain a water-containing porous material, and the water-containing porous material is freeze-dried at -40 °C to obtain a degradable sponge body.

[0073] Example 4

[0074] The preparation method of the polyethylene glycol-polylysine-polyvaline triblock copolymer refers to the method disclosed in Patent 201610345388.8. Sodium alginate is commercially available medical sodium alginate, and gelatin is commercially available medical gelatin.

[0075] The mass ratio of the usage amounts of gelatin, modified sodium alginate and polyethylene glycol-polylysine-polyvaline triblock copolymer is 5:3:3. Before use, the raw materials are washed 3 times with distilled water. The above three raw material substances are stirred and mixed evenly to prepare a mixed material. Water with a weight portion 5 times that of the mass of the mixed material is added, stirred evenly, injected into a mold and then vacuum-sealed, and crosslinked by electron beam radiation at 30 kGy to obtain a hydrogel; the hydrogel is soaked in sterile water for 36 h until swelling equilibrium is reached to obtain a water-containing porous material, and the water-containing porous material is freeze-dried at -40 °C to obtain a degradable sponge body.

[0076] Example 5

[0077] The preparation method of the polyethylene glycol-polylysine-polyvaline triblock copolymer refers to the method disclosed in Patent 201610345388.8. Gelatin is commercially available medical gelatin.

[0078] The mass ratio of the usage amounts of gelatin and polyethylene glycol-polylysine-polyvaline triblock copolymer is 5:6. Before use, the raw materials are washed 3 times with distilled water. The above two raw material substances are stirred and mixed evenly to prepare a mixed material. Water with a weight portion 5 times that of the mass of the mixed material is added, stirred evenly, injected into a mold and then vacuum-sealed, and crosslinked by electron beam radiation at 30 kGy to obtain a hydrogel; the hydrogel is soaked in sterile water for 36 h until swelling equilibrium is reached to obtain a water-containing porous material, and the water-containing porous material is freeze-dried at -40 °C to obtain a degradable sponge body.

[0079] Example 6

[0080] Cutting and homogenization treatment of the patch material: In a clean bench, take the degradable sponge prepared in Examples 2-5, and use an 8 mm round punch to cut the material; the cut material is a cylinder with a diameter of 8 mm and a thickness of 5 mm. Without weighing, the mass difference between each sponge sample can be ensured to be within 0.0004 g, and no secondary sterilization is required.

[0081] Porosity determination of the patch material: Place anhydrous ethanol in a graduated cylinder, and record the volume as V1; immerse the patch material and let it be completely soaked, and record the volume as V2; take out the patch material, and record the remaining volume as V3; the porosity calculation formula of the patch material is δ=(V1-V3) / (V2-V3)×100%; for the above determination, take 3 samples, calculate the average value and standard deviation, and the porosity of the patch material is shown in Table 1:

[0082] Table 1

[0083]

[0084] Note: * represents P<0.05 compared with the group of Example 2.

[0085] As can be seen from Table 1, the porosities of the groups of Examples 3-5 are significantly lower than that of Example 2.

[0086] Water absorption determination of the patch material: The water absorption of the patch material is determined by the weighing method. The dry weight of the material is recorded as W1. The material is immersed in ultrapure water for 24 h and completely soaked. Then take out the patch material, remove the surface moisture and weigh it, and record it as W2; the calculation formula is: θ=[(W2-W1) / W2]×100%; for the above determination, take 3 samples, calculate the average value and standard deviation, and the water absorption of the patch material is shown in Table 2:

[0087] Table 2

[0088]

[0089] As can be seen from Table 2, the water absorptions of the groups of Examples 2-5 are all relatively high, and there is no significant difference between the groups.

[0090] Preparation and performance investigation of the stem cell preparation sponge patch complex:

[0091] Determination of the method for loading cells onto the material: Take a cylindrical sponge patch with a diameter of 8 mm and a thickness of 5 mm after cutting (the sponge patch material is prepared in Example 2), place it in a 24-well culture plate, and pre-soak it with a culture medium (the culture medium is divided into 4 groups. The first group is a common neural stem cell culture medium, the second group is a common neural stem cell culture medium added with L-carnitine (final concentration 4 μg / mL), the third group is a common neural stem cell culture medium added with vitamin E (final concentration 4 μg / mL), and the fourth group is a common neural stem cell culture medium added with L-carnitine (final concentration 1 μg / mL) and vitamin E (final concentration 3 μg / mL)); Take the prepared single-cell suspension of neural stem cells and inoculate 250 μL per well, and apply it after incubation for 1 h. The following inoculation of cells onto the material is carried out in this way.

[0092] Determination of the cell-loading capacity of the material: The cells are inoculated onto the material. The cell densities of the first to fourth groups are set at 4×10 7 cells / mL; Take out the sponge, add saline to collect the cells that did not adhere to the sponge during the cell inoculation process; Count the remaining liquid volume and cell concentration, calculate the remaining cell number; According to the adsorption rate = adsorbed cell number / original cell number, the cell adsorption rates of the first to fourth groups are calculated as: 97.8%, 98.5%, 97.5%, 97.8%.

[0093] Detection of the biocompatibility of the material using the material extract: Take a cylindrical sponge patch with a diameter of 8 mm and a thickness of 5 mm after cutting (the sponge patch material is prepared in Examples 2-5), put it into a culture flask, add the neural stem cell culture medium of the first to fourth groups, and place it in an incubator at 37°C for 24 h to prepare a sponge patch extract; Prepare culture media containing material extract concentrations of 100%, 50%, 25%, and 0% respectively, and store them refrigerated at 4°C for later use; Inoculate the cells into a 96-well plate at a density of 4×10 7 cells / mL, and add the above 4 kinds of concentration material extracts respectively, and place them in an incubator at 37°C and 5% CO2 for culture. The experimental groups are set as 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, and 14 days groups; At the set time points, first discard the original culture medium and then add 100 μL of the corresponding culture medium and 10 μL of CCK-8 solution, place it in the incubator for incubation for 2 h, and then measure the absorbance with an enzyme-linked immunosorbent assay instrument. The growth curve results obtained by CCK-8 detection show that the extracts of different groups have no effect on the proliferation ability of cells.

[0094] The sponge patch material is prepared in Examples 2-5. The neural stem cell culture medium is a neural stem cell culture medium added with L-carnitine (final concentration 1 μg / mL) and vitamin E (final concentration 3 μg / mL). Select a sponge patch complex sample with neural stem cells cultured for 4 days, and the cell inoculation density is 4×10 7cells / mL; Discard the original culture medium, wash twice with PBS buffer, add Live / Dead stain (Calcein-AM / PI) to cover the material, place it in an incubator at 37°C and 5% CO2 for half an hour, observe and take pictures under a confocal microscope. The fluorescence represents the cell viability staining. Count the survival of the cells and calculate the survival rate:

[0095] Survival rate = number of live cells / (number of live cells + number of dead cells) × 100%

[0096] The measurement results are shown in Table 3:

[0097] Table 3

[0098]

[0099] Note: * represents P < 0.05 compared with the group of Example 2.

[0100] The sponge patch material was prepared according to Example 2. The neural stem cell culture medium was divided into 4 groups. The first group was the ordinary neural stem cell culture medium, the second group was the ordinary neural stem cell culture medium added with L-carnitine (final concentration 4 μg / mL), the third group was the ordinary neural stem cell culture medium added with vitamin E (final concentration 4 μg / mL), and the fourth group was the ordinary neural stem cell culture medium added with L-carnitine (final concentration 1 μg / mL) and vitamin E (final concentration 3 μg / mL). Select the sponge patch complex samples cultured with neural stem cells for 4 days, and the cell seeding density was 4 × 10 7 cells / mL; Discard the original culture medium, wash twice with PBS buffer, add Live / Dead stain (Calcein-AM / PI) to cover the material, place it in an incubator at 37°C and 5% CO2 for half an hour, observe and take pictures under a confocal microscope. The fluorescence represents the cell viability staining. Count the survival of the cells and calculate the survival rate:

[0101] Survival rate = number of live cells / (number of live cells + number of dead cells) × 100%

[0102] The measurement results are shown in Table 4:

[0103] Table 4

[0104]

[0105] Note: * represents P < 0.05 compared with the group of Example 2, and * represents P < 0.01.

[0106] Investigation of in vitro degradation of the patch material: Take 21 samples prepared according to Example 2 and inoculate 4 × 10 7Neural stem cells with a density of cells / mL were cultured continuously; 3 samples were taken at each time point at 0 hour, 6 hours, 1 day, 3 days, 7 days, 14 days, and 21 days; after washing three times with ultrapure water, the samples were dried at 40°C and weighed; the degree of degradation of the biomaterial was determined by calculating the residual weight. Under the action of cells, the mass of the sponge material was greatly improved compared with the original mass at 8 hours, which was attributed to the swelling of the material; the mass subsequently decreased, and on the 22nd day, the sample was in a gel-like state, which was completely dissolved after washing, achieving complete degradation.

[0107] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A stem cell preparation sponge patch complex, characterized in that, The sponge patch complex is a degradable sponge body infiltrated with a stem cell suspension, and the degradable sponge body is made of gelatin, modified sodium alginate, and poly(ethylene glycol)-poly(L-lysine)-poly(L-valine) triblock copolymer; the mass ratio of the gelatin, modified sodium alginate, and poly(ethylene glycol)-poly(L-lysine)-poly(L-valine) triblock copolymer is 5:(1 - 6):(1 - 6); The modified sodium alginate is prepared by the following method: Dissolve sodium alginate in an aqueous solution with a mass 15 - 30 times that of sodium alginate, adjust the pH to 3 - 5, add N-hydroxysuccinimide, stir at 20 - 30 °C for 1 - 3 h, then add L-cysteine methyl ester hydrochloride, raise the temperature to 50 - 70 °C, continue stirring and reacting for 1 - 3 h, stop stirring, carry out sedimentation with ethanol, filter and collect the precipitate, wash the precipitate with ethanol 2 - 4 times, and vacuum dry for 3 - 5 h to obtain the modified sodium alginate; the molar ratio of the carboxylate group in the sodium alginate, N-hydroxysuccinimide, and L-cysteine methyl ester hydrochloride is 3:(0.5 - 3):(0.5 - 3).

2. The stem cell preparation sponge patch complex according to claim 1, wherein The stem cells are neural stem cells.

3. The stem cell preparation sponge patch complex according to claim 1, wherein The concentration of the stem cell suspension is 8×10 6 -4×10 7 cells / mL.

4. The stem cell preparation sponge patch complex according to claim 1, characterized in that The porosity of the degradable sponge body is more than 85%.

5. The preparation method of the stem cell preparation sponge patch complex according to claim 1, characterized in that, It includes the following steps: pre-soak a degradable sponge with a culture medium, then take a pre-prepared single-cell suspension of neural stem cells, dilute the suspension to a cell density of 8×10 6 -4×10 7 cells / mL, and fully infiltrate the degradable sponge with it until it is saturated with absorption.

6. The preparation method of the stem cell preparation sponge patch complex according to claim 5, characterized in that, The culture medium is a complete culture medium for neural stem cells, and 1 μg / mL of L-carnitine and 3 μg / mL of vitamin E are added to the culture medium.

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