CN115252539A - Polypeptide hydrogel for loading stem cells and preparation method and application thereof - Google Patents
Polypeptide hydrogel for loading stem cells and preparation method and application thereof Download PDFInfo
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- CN115252539A CN115252539A CN202210878226.6A CN202210878226A CN115252539A CN 115252539 A CN115252539 A CN 115252539A CN 202210878226 A CN202210878226 A CN 202210878226A CN 115252539 A CN115252539 A CN 115252539A
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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Abstract
The invention discloses a polypeptide hydrogel for loading stem cells, and a preparation method and application thereof. The polypeptide hydrogel comprises: the polypeptide A, the polypeptide B, the platelet-rich plasma and the placental amnion extract, wherein the primary structural formula of the self-assembled polypeptide comprises a formula (1) or a formula (2), and the primary structural formula of the polypeptide B comprises a formula (3). The polypeptide hydrogel for loading stem cells has the bionic property of extracellular matrixes, is favorable for cell adhesion and growth, can effectively avoid cell damage, contains rich growth factors, effectively maintains the activity of cells, can obviously improve the survival rate of MenSCs in vitro, does not need resuscitation when in use, is convenient to store, transport and clinically popularize, has good biocompatibility, can improve the retention time of MenSCs in uterus, and has obvious treatment effect on female IUA diseases.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to a polypeptide hydrogel for loading stem cells, and a preparation method and application thereof.
Background
Intrauterine adhesion (IUA), also known as Asherman syndrome, refers to a series of syndromes caused by the serious damage of the endometrial basement layer due to traumatic operation and infection of the uterine cavity, the formation of fibrosis scar and the partial or total blockage of the uterine cavity, including the serious influence on the female reproductive function. At present, the resection of the intrauterine adhesion is the preferred scheme for treating the intrauterine adhesion, however, patients with severe intrauterine adhesion have high recurrence rate after treatment and low pregnancy rate, and the fertility is difficult to be improved fundamentally.
Menses-derived mesenchymal stem cells (MenSCs) are novel adult mesenchymal stem cells which are separated and cultured from female menstrual blood, have the outstanding advantages of rich sample sources, noninvasive material taking, low immunogenicity and no ethical problems, are ideal stem cell sources for regenerative medicine research and clinical transformation, and are widely used for the treatment research of diseases of various tissues and organs such as muscles, skin, heart, liver, brain, ovary, uterus and the like. Research finds that MenSCs intrauterine transplantation can effectively promote endometrial regeneration and uterine cavity morphological restoration of IUA patients, the clinical pregnancy rate and The survival rate after treatment are obviously superior to those of conventional treatment means, and The clinical application is safe and efficient (automatic mental volume-derived structural cells transplantation for segment assistant's syndrome [ J ]. Hum repeat.2016; the multi-functional rolls of mental volume-derived structural cells in regenerative medicine [ J ]. Stem Cell Ther.2019), thereby providing a new treatment strategy for refractory infertility of uterine cavity adhesion.
Currently, there are two methods of cell preservation used in the technology of stem cell transplantation: cryopreservation in liquid nitrogen and preparation of cell suspension by physiological saline cryopreservation (4 ℃). The liquid nitrogen is stored at low temperature for a long time, but real-time temperature monitoring is needed in the process of cell storage and transportation, and the cell is rapidly inactivated due to out-of-control temperature; the stem cells stored in the liquid nitrogen need to be thawed and revived before being transplanted, the activity of the cells is detected again, the operation is complicated, and the clinical application is not easy to realize. The MenSCs suspension prepared from physiological saline and human albumin can be directly used for intrauterine perfusion therapy, but the storage time of the stem cell preparation is short, the cell activity can be maintained for 12h (more than or equal to 90%) at low temperature (4 ℃), and the cell activity rapidly decreases after 24 h. In addition, the current MenSCs adopt a cell suspension uterine cavity perfusion administration mode for treating the uterine cavity adhesion, and the problems of short cell residence time and unstable treatment effect exist in local administration.
In summary, how to provide a method for preserving a mesenchymal stem cell from blood, which is simple and convenient to operate, has a good preservation effect and is beneficial to drug administration, is one of the problems to be solved in the field of application of mesenchymal stem cells from blood.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a polypeptide hydrogel for loading menstrual blood stem cells and a preparation method and application thereof, wherein the polypeptide hydrogel has the bionic characteristic of simulating extracellular matrix, is beneficial to cell adhesion and growth, can avoid cell damage caused by violent shaking in transportation, is rich in growth factors, effectively maintains the activity of cells, has simple preparation process and low cost, is easy to amplify and produce, and is suitable for clinical application.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a polypeptide hydrogel for loading menstrual blood stem cells, the polypeptide hydrogel comprising: the polypeptide A comprises self-assembly polypeptide, the primary structural formula of the self-assembly polypeptide comprises a formula (1) or a formula (2), and the primary structural formula of the polypeptide B comprises a formula (3).
The polypeptide hydrogel is prepared by combining the polypeptide, the platelet-rich plasma and the placental amniotic membrane extract, has the bionic characteristic similar to that of extracellular matrix, is favorable for cell adhesion and growth, has good hydrophilicity, can avoid cell damage caused by severe shaking in transportation, contains rich collagen, fibronectin, laminin, elastin, proteoglycan, hyaluronic acid, basic fibroblast growth factor, transforming growth factor and the like, can effectively maintain the cell activity, has high safety and good biocompatibility, and can be effectively applied to cell preservation.
Formula (1): ac- (Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala)n-NH2Wherein n is an integer of 2 to 8.
Formula (2): ac- (Lys-Ala-Glu-Ala-Lys-Ala-Glu-Ala)n-NH2Wherein n is an integer of 2 to 8.
In the invention, ac is N-terminal acetylation NH2Is C-terminal amidation; the polypeptide A is formed by alternating hydrophilic amino acids and hydrophobic amino acids, wherein Arg and Lys are positively charged hydrophilic amino acids, asp and Glu are negatively charged hydrophilic amino acids, and Ala is hydrophobic amino acid.
In the specific embodiment of the invention, the number n of the amino acid sequence repeats in the sequence can be 2, so that the ion complementary polypeptide consisting of 16 amino acids is formed, the intermolecular interaction of the polypeptides is firmer, and a more stable and ordered self-assembly structure is formed.
Formula (3): arg-Gly-Asp.
Preferably, the concentration of the polypeptide A in the polypeptide hydrogel is 0.28-1.60 mg/mL, including but not limited to 0.29mg/mL, 0.30mg/mL, 0.35mg/mL, 0.38mg/mL, 0.4mg/mL, 0.9mg/mL, 1mg/mL, 1.2mg/mL, 1.3mg/mL, 1.4mg/mL or 1.5mg/mL.
Preferably, the concentration of the polypeptide B in the polypeptide hydrogel is 0.07-0.4 mg/mL,0.08mg/mL, 0.09mg/mL, 0.1mg/mL, 0.2mg/mL or 0.3mg/mL.
Preferably, the volume ratio of the platelet rich plasma in the polypeptide hydrogel is 2% to 10% (e.g., 3%, 4%, 5%, 6%, 7%, 8% or 9%), and the platelet rich plasma contains 1 × 10%9/L～30×109Platelets at/L concentration, including but not limited to 2X 109/L、4×109/L、8×109/L、10×109/L、12×109/L、15×109/L、18×109/L、25×109/L、26×109/L、27×109Or 28X 109/L。
Preferably, the concentration of the placental membrane extract in the polypeptide hydrogel is 2.5-4 mg/mL, including but not limited to 2.6mg/mL, 2.7mg/mL, 2.8mg/mL, 3mg/mL, 3.2mg/mL, 3.4mg/mL, 3.6mg/mL, 3.8mg/mL or 3.9mg/mL.
In the present invention, platelet-rich plasma products are commercially available, such as human platelet lysates; commercially available placental amniotic membrane extracts or amniotic homogenate, or platelet rich plasma and placental amniotic membrane extracts extracted using methods commonly used in the art are suitable for use in the present invention.
In the present invention, platelet-rich plasma (PRP) can be prepared according to the literature (Effects of platelet-rich plasma on the activity of human mesenchymal blood-derived fetal cells in vitro. Stem Cell Res The 2018), and the preparation method of the placental amniotic membrane extract can comprise the following steps:
(1) Washing placenta amnion with normal saline, and scraping sponge layer of amnion;
(2) Treating with 0.5-2% Triton for 8-16 h, and soaking in deionized water for 10-60 min;
(3) Treating with 0.1-0.4% EDTA for 5-12 hr;
(4) Treating for 1-6 h by using DNase enzyme, transferring to PBS, and standing for 1-2 h;
(5) Washing with deionized water and drying to obtain acellular amnion;
(6) Weighing a proper amount of acellular amniotic membrane before use, and soaking the amniotic membrane in physiological saline for 48-96 h to reach the final concentration of 2-20 mg/mL;
(7) Homogenizing for 2-3 times at 1-2 ten thousand rpm by using a homogenizer, and centrifuging for 1-2 min;
(8) Centrifuging the obtained homogenate for 30-40 min at 500-600 Xg;
(9) And filtering the supernatant, and adjusting the pH to 7-7.5 to obtain the placental amnion extract.
In the present invention, the placental amniotic membrane extract may be derived from human placental amniotic membrane.
In a second aspect, the present invention provides a method for preparing a polypeptide hydrogel for loading with menstrual blood stem cells according to the first aspect, the method comprising:
and mixing the solution of the polypeptide A and the solution of the polypeptide B to obtain a mixed solution, and mixing the mixed solution with the platelet-rich plasma and the placental amnion extract to obtain the polypeptide hydrogel.
In a third aspect, the invention provides the use of the polypeptide hydrogel for loading with menstrual blood stem cells according to the first aspect in cell preservation.
According to the invention, the polypeptide hydrogel has the bionic characteristics of an extracellular matrix, is beneficial to cell adhesion and growth, effectively avoids cell damage, is rich in growth factors, can effectively maintain cell activity, can effectively preserve cells, is theoretically applicable to any living animal cell, and is not specially limited.
In the present invention, the cell may be selected from any one of or a combination of at least two of a menstrual blood-derived mesenchymal stem cell, a bone marrow mesenchymal stem cell, an umbilical cord mesenchymal stem cell, an embryonic stem cell, a placental mesenchymal stem cell, an endometrial stem cell, or an induced pluripotent stem cell.
In a fourth aspect, the present invention provides the use of the polypeptide hydrogel for loading with menstrual blood stem cells according to the first aspect in the preparation of a cell preparation.
In the present invention, the cell may be selected from any one of or a combination of at least two of the cells including a mesenchymal stem cell of a blood source, a mesenchymal stem cell of a bone marrow, a mesenchymal stem cell of an umbilical cord, an embryonic stem cell, a mesenchymal stem cell of a placenta, an endometrial stem cell or an induced pluripotent stem cell.
In a fifth aspect, the present invention provides a cell preparation comprising the polypeptide hydrogel for loading with menstrual blood stem cells of the first aspect and live cells.
In the present invention, the cells may be selected from any one of or a combination of at least two of the living cells including mesenchymal stem cells of menstrual origin, mesenchymal stem cells of bone marrow, mesenchymal stem cells of umbilical cord, embryonic stem cells, mesenchymal stem cells of placenta, endometrial stem cells or induced pluripotent stem cells.
Preferably, the number of the generations of the living cells in the preparation is 1 × 10, and the generations of the living cells in the preparation are P2-P66～5×106And (4) respectively.
Preferably, the cell preparation further comprises any one or a combination of at least two of a preservative, an antioxidant and an antimicrobial.
Preferably, the antioxidant comprises any one or a combination of at least two of a prebiotic, a tea polyphenol, vitamin C or vitamin E.
Preferably, the antibacterial agent comprises any one of carbomer, chitosan or nano-silver or a combination of at least two of the same.
In a sixth aspect, the present invention provides a method for preparing a cell preparation according to the fifth aspect, the method comprising:
mixing the solution of the polypeptide A and the solution of the polypeptide B to obtain gel mother liquor, diluting the gel mother liquor to obtain gel working solution, mixing platelet-rich plasma, an antioxidant, an antibacterial agent and living cells to obtain cell mixed solution, and mixing the gel working solution, the cell mixed solution and the placenta amnion extract to obtain the cell preparation.
Preferably, the concentration of the polypeptide A and the polypeptide B in the gel working solution is 0.56 to 3.20mg/mL (e.g., 0.58mg/mL, 0.6mg/mL, 0.9mg/mL, 1mg/mL, 1.2mg/mL, 1.5mg/mL, 1.8mg/mL, 2mg/mL, 2.5mg/mL, 2.6mg/mL, 2.8mg/mL, 3mg/mL, or 3.1 mg/mL), and 0.14 to 0.80mg/mL (e.g., 0.15mg/mL, 0.16mg/mL, 0.18mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.65mg/mL, 0.7mg/mL, 0.72mg/mL, 0.76mg/mL, or 0.78 mg/mL), respectively.
Preferably, the number of viable cells in the cell mixture is 1X 106～5×106And (4) respectively.
Preferably, the volume percentage of the platelet-rich plasma in the cell mixture is 2-10%, wherein the volume percentage comprises 1 × 109/L～30×109Platelets at/L concentration.
Preferably, the volume ratio of the cell mixture, the placental amniotic membrane extract and the gel working solution is 1.
In a seventh aspect, the present invention provides the use of the polypeptide hydrogel for loading with a hematopoietic stem cell of the first aspect or the cell preparation of the fifth aspect in the preparation of a stem cell therapeutic agent.
In the present invention, the cells may be selected from any one of or a combination of at least two of the living cells including a menstrual blood-derived mesenchymal stem cell, a bone marrow mesenchymal stem cell, an umbilical cord mesenchymal stem cell, an embryonic stem cell, a placental mesenchymal stem cell, an endometrial stem cell, or an induced pluripotent stem cell.
The polypeptide hydrogel for loading the menstrual blood stem cells has the bionic property of extracellular matrixes, is beneficial to cell adhesion and growth, can effectively protect cells and maintain the activity of the cells, has good biocompatibility, can be used as an effective drug carrier, such as a stem cell drug, for example, a novel stem cell therapeutic drug slow-release support is provided for uterine cavity adhesion prevention and treatment, can effectively improve the survival rate of the menstrual blood-derived mesenchymal stem cells (MenSCs) in vitro, can be stored for 7 days at 4 ℃, has the cell activity rate still exceeding 90%, does not need to be recovered when in use, has good biocompatibility, can improve the residence time of the MenSCs in uterus, has an obvious treatment effect on female IUA diseases, and is convenient to store, transport and clinically popularize.
In an eighth aspect, the present invention provides a stem cell therapeutic drug comprising the cell preparation of the fifth aspect.
Preferably, the stem cell therapeutic drug further comprises an adjuvant.
Preferably, the adjuvant comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, a diluent, a preservative, an antioxidant, an antibacterial agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant or a pH regulator.
Compared with the prior art, the invention has the following beneficial effects:
(1) The polypeptide hydrogel loaded with the menstrual blood stem cells has the bionic property similar to that of extracellular matrix, is beneficial to cell adhesion and growth, has good hydrophilicity, can avoid cell damage caused by severe shaking in transportation, contains abundant collagen, fibronectin, laminin, elastin, proteoglycan, hyaluronic acid, basic fibroblast growth factor, transforming growth factor and the like, effectively maintains the cell activity, has simple preparation process and low cost, is easy to amplify and produce, and is suitable for clinical application;
(2) The polypeptide hydrogel prepared by the invention can obviously improve the survival rate of MenSCs in vitro, the cell survival rate still exceeds 90 percent after being stored for 7 days at 4 ℃, resuscitation is not needed during use, the storage, transportation and clinical popularization are convenient, the safety is high, meanwhile, the polypeptide hydrogel has good biocompatibility, the residence time of MenSCs in uterus can be prolonged, and the polypeptide hydrogel has an obvious treatment effect on female IUA diseases.
Drawings
FIG. 1 is a drawing of a culture of a blood-derived mesenchymal stem cell, with a scale bar of 100 μm;
FIG. 2A is a graph of the results of the phenotype (CD 90, CD44, CD73, CD 105) identification of mesenchymal stem cells of blood origin;
FIG. 2B is a graph showing the results of the phenotype (CD 45, CD38, CD11B, HLA-DR, CD19, CD 34) identification of mesenchymal stem cells of blood origin;
FIG. 3 is a diagram of a polypeptide hydrogel cell preparation;
FIG. 4 is a graph of elastic modulus of different polypeptide hydrogel cell preparations;
FIG. 5 is a graph showing the results of cell viability for 7 days in the AGM, GM and SM groups in example 3;
FIG. 6 is a graph showing the results of 17 days-old cell viability change of the AGM, GM and SM groups in example 3;
FIG. 7 is a graph showing the results of population doubling times for P3 generation of fresh MenSCs cells and the AGM group of example 3 storing MenSCs cells at 7 d;
FIG. 8 is a graph showing the cell cycle results of P3 generation fresh MenSCs cells;
FIG. 9 is a graph showing the results of cell cycle of the AGM group storing MenSCs cells of 7d in example 3.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
In the present embodiment, the effect of the polypeptide gel of the present invention was verified by taking mesenchymal stem cells of blood origin (MenSCs) as an example.
Example 1
This example performed isolated culture and identification of mesenchymal stem cells derived from blood.
1. Preparation of autologous serum
Collecting peripheral blood of a patient with intrauterine adhesion by a venous blood collection method, collecting the total amount of about 20mL, standing the collected blood sample in a refrigerator at 4 ℃ for 12h, centrifuging at 3000rpm for 10min, taking upper serum, placing the serum in a constant-temperature water bath at 56 ℃, inactivating for 30min, and storing the obtained serum in the refrigerator at-20 ℃ for later use.
2. Isolated culture of mesenchymal stem cells of menstrual blood source
Sterile menstrual blood samples were collected by using a menstrual cup, and mononuclear cells were isolated by using a sample density separation medium (Shenzhenjn Dake, 7912011) within 48h, and the detailed steps refer to the specification of the use of the separation medium. The resulting cell pellets were resuspended in complete medium (DMEM/F12 medium containing 10% autologous serum), inoculated into a flask and placed at 5% CO2Culturing in an incubator with saturated humidity at 37 ℃, completely replacing 5mL of completely-prepared culture solution every 2 days, observing under a microscope in the culture process, as shown in figure 1, showing that the mesenchymal stem cells from blood sources become short rods or spindle type, have longer protrusions, are bright and three-dimensional, replacing the culture solution for three days, and culturing and amplifying to P3 generation.
3. Phenotypic identification of mesenchymal stem cells of menstrual origin
By substituting P3 for 5X 105Marking antibodies of CD34, CD38, CD45, CD19, CD11B, HLA-DR, CD44, CD73, CD90 and CD105 by cell suspension, incubating for 30min in a refrigerator at 4 ℃ in a dark place, washing out unbound antibodies by PBS, taking corresponding isotype control antibodies as a negative control group, detecting and analyzing by using a Cytoflex flow cytometer (Beckman), and identifying cell phenotypes by using CytExpert software, wherein the results show that the CD90, the CD44, the CD105 and the CD73 are all more than or equal to 95 percent, the CD34, the CD38, the CD45, the CD19, the CD11B and the HLA-DR are all less than or equal to 2 percent, and the cell line is identified to be mesenchymal stem cells.
Example 2
This example prepared polypeptide hydrogels and stem cell preparations.
1. Preparation of gel working solution
(1) Self-assembled short peptide (Ac-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-NH)2) Dissolving 10mg of lyophilized powder (AbMole, M10196) in 1mL of sterile deionized water, and performing ultrasonic treatment for 30min at 37 ℃ by using an ultrasonic cleaner to fully dissolve the lyophilized powder to obtain a polypeptide mother solution A with the concentration of 10mg/mL (M/v);
(2) Dissolving 10mg (Sigma, A8052) of polypeptide B (Arg-Gly-Asp) freeze-dried powder in 1mL of sterile deionized water, and carrying out ultrasonic treatment for 30min by using an ultrasonic cleaner at 37 ℃ for full dissolution to obtain polypeptide mother liquor B with the concentration of 10mg/mL (m/v);
(3) Uniformly mixing the polypeptide mother liquor A and the polypeptide mother liquor B according to a volume ratio of 4;
(4) Adjusting the pH value of the mixed hydrogel mother liquor to 7.4, wherein the prepared hydrogel mother liquor can be stored at 4 ℃, and when in use, the prepared hydrogel mother liquor is diluted by deionized water to 1.
2. Preparing placental amnion extract
(1) Aseptically stripping placenta amnion, repeatedly washing with normal saline, and scraping surface mucus and residual bloodstain;
(2) Performing blunt dissection, and scraping off the sponge layer of the amnion to obtain smooth and semitransparent amnion.
(3) 1% Triton treatment for 12h, soaking in deionized water for 30min;
(4) 0.25% EDTA treatment for 8h;
(5) Treating with DNase for 4h, transferring to PBS, and standing for 1h;
(6) Repeatedly cleaning in deionized water for 3 times, placing into a freeze dryer for 8h, sterilizing with ethylene oxide, and storing at-80 deg.C;
(7) Before use, shearing a proper amount of acellular amniotic membrane, weighing, and soaking the amniotic membrane in normal saline for 72 hours to reach the final concentration of 10 mg/mL;
(8) Homogenizing with homogenizer (HTY-761) at 2 ten thousand rpm for 3 times (1 min);
(9) The obtained homogenate was centrifuged at 600 Xg for 30min;
(10) Filtering the supernatant with 0.22 μm filter, adjusting pH to 7.2 to obtain placenta amnion extract, and storing in refrigerator at 4 deg.C.
3. Preparation of autologous platelet-rich plasma (PRP)
Reference literature for preparation method of PRP (Effects of platelet-rich plasma on the activity of human physiological plasma in vitro. Stem Cell Res The.2018), extracting from single-sampling plateletTaking PRP (concentration of 12.5X 10)11(ii)/L, for activation, adding 20% CaCl in a volume ratio of 12(Sigma-Aldrich) and 1000U/mL thrombin (T8020, solarbio), the mixture was incubated at 37 ℃ for 1h, then at 4 ℃ for 12h, the gelatinous mixture was centrifuged at 5000rpm for 30min at 4 ℃ in order to collect the activated PRP, the supernatant aspirated and filtered through a 0.22 μm filter, then portioned and stored at-80 ℃ to avoid repeated freezing and thawing.
And mixing the working solution, the placental amnion extract and the PRP according to the requirement to prepare the polypeptide hydrogel.
4. Preparation of cell suspensions
MenSCs of P3 were collected with a cell count of 1X 106The cells were suspended in 110. Mu.L of physiological saline at 88%, then 10% PRP was added to provide growth factors and cytokines, 1% of prebiotic polyphenol was added as an antioxidant, and 1% of carbomer was added as an antibacterial and anti-inflammatory substance.
5. Preparation of polypeptide hydrogel cell preparation
Adding the amnion extract and the hydrogel working solution into the cell suspension in sequence at a volume ratio of 12And (3) preparing a formed gel preparation in a cell incubator for 30min, inverting the formed gel preparation, adhering the formed gel preparation to a penicillin bottle without flowing down, and as shown in fig. 3, when the hydrogel mother liquor is diluted into a working solution by adopting different proportions, the elastic modulus (G') of the final gel preparation is different, and the elastic modulus of the gel preparation is tested by using a dynamic rheometer, as shown in fig. 4, when the proportion of the hydrogel working solution is 1.
Example 3
This example tests the survival rate of cells preserved by polypeptide hydrogel.
Comparing the polypeptide hydrogel prepared by the invention with the polypeptide hydrogel without the placental amniotic membrane extract (the preparation method is as in example 2, except that the amniotic membrane extract component is not added, and the normal saline is replaced in equal proportion, namely the normal saline and the hydrogel working solution are sequentially added into the cell suspension, the volume ratio is 1,well mixed to trigger cross-linking) and normal saline plus albumin preserved stem cell activity. Experimental group 1 is 500. Mu.L of the polypeptide hydrogel cell preparation of the present invention containing 1X 106MenSCs, represented by AGM group; experimental group 2 was a polypeptide hydrogel cell preparation containing no placental amniotic membrane extract, and similarly 500. Mu.L of the same was 1X 106MenSCs, expressed as GM group; the control group was a normal saline-albumin preparation 500. Mu.L containing 1X 106Each MenSCs, represented by SM group, was set to 3 sample replicates, temperature simulated clinical transport 4 ℃, retention time groups were 13 groups, respectively: 0.5d (day), 1d, 1.5d, 2d, 3d, 4d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, and the cell viability was counted using trypan blue staining after centrifugation to recover cells, according to the formula: (number of normal cells/total cells) × 100%, statistical analysis of cell viability was performed using GraphPad Prism 8, statistical analysis of multiplex comparisons between groups was performed using one-way analysis of variance, and p is expressed as<0.05, represents p<0.01, represents p<0.001 is considered statistically significant and the results of the study show that: when the stem cell preparation is placed for 0.5d, the activity of the three groups is not different; when the cells are placed for 2d, the activity of the cells in the SM group is reduced by half, and when the cells are placed to 7d, all the cells in the SM group die; when the AGM group and the GM group are placed for more than 5 days, a significant difference begins to appear (p)<0.001 ); when placed at 7d, the AGM group had a cell viability of over 90% and was still available for clinical uterine perfusion, whereas the GM group had a cell viability reduced to 70% and failed to meet the clinical use criteria (see FIG. 5). In addition, as shown in fig. 6, the MenSCs survival rate preserved in AGM group was significantly higher than that of GM group with the increase of preservation time, indicating that the gel preparation containing placental amniotic membrane extract can significantly improve the bioactivity of instant menstrual blood stem cells.
Example 4
This example measures the cell population doubling time of a polypeptide hydrogel cell preparation.
P3 generation of fresh MenSCs cells and 7d cells stored in AGM group of example 3 were collected at 1X 104The density of/mL, 1mL of each well is inoculated to a 24-well plate, the plate is placed in an incubator, the total liquid change is carried out every 3 days, 3-well cells are digested and counted after 24 hours, the digestion and counting of the cells are repeated every day within 1-8 days, and 8 days are carried outAnd then drawing a growth curve, calculating the population doubling time according to a pharmacopoeia method, and calculating the population doubling time after repeating the three times, wherein the detection result of the population doubling time is shown in table 1, the cell growth curve is shown in a representative graph of fig. 7, compared with the fresh cells, the population doubling time of the AGM group gel preparation cells at the 7 th day has no significant difference (P is more than 0.05), and the invention shows that the cells can be effectively maintained by using the polypeptide hydrogel for storing the cells, and the doubling activity after storing for 7 days is similar to that of the fresh cells.
TABLE 1
| Number of repetitions | Fresh cells (h) | |
| 1 | 31.0 | 31.5 |
| 2 | 33.6 | 34.0 |
| 3 | 32.1 | 32.9 |
| Mean value of | 32.23 | 32.8 |
Example 5
This example examined the cell cycle of polypeptide hydrogel cell preparations.
Collection of P3 generation fresh MenSCs cells and preservation of 7d cells in the AGM group of example 3, each at 1X 106Resuspending the cells with 0.5mL of precooled PBS, adding 1.2mL of precooled absolute ethanol (final ethanol concentration: 70%), blowing and mixing uniformly to avoid cell aggregation, fixing overnight at 4 ℃, centrifuging (300 Xg, 5 min) to collect the fixed cells, washing the cells once with PBS, adding 500. Mu.L of PI staining agent into the cell sediment, collecting and detecting 3 thousands of cells by a flow cytometer, and analyzing the result by ModFit FL fitting, and calculating the formula according to Proliferation Index (PI): PI = (S + G2/M)/(G0/G1 + S + G2/M) calculation, cell cycle detection results are shown in Table 2 after repeating for three times, cell cycle fitting analysis representative graphs of 7 th days of fresh cells and AGM groups are respectively shown in FIGS. 8 and 9, wherein an RCS value of less than 3.0 indicates that the fitted curve is good in matching degree with real results, a% CV of less than 8.0% indicates that the ratio of S phases is accurate, and Debris and Aggregates of less than 20% indicate that fragments and adherends are normal, and results of repeating for three times show that proliferation indexes of fresh cells and 7d cells stored in AGM are not significantly different (P is more than 0.05), and the storage effect of the AGM group on 7 th day is good.
TABLE 2
| Number of repetitions | Fresh cells | AGM preservation of |
| 1 | 26.38 | 25.30 |
| 2 | 26.82 | 25.53 |
| 3 | 27.60 | 26.92 |
| Mean value of | 26.93 | 25.92 |
In conclusion, the polypeptide hydrogel disclosed by the invention has the bionic property similar to that of an extracellular matrix, is favorable for cell adhesion and growth, has good hydrophilicity, can avoid cell damage caused by severe shaking in transportation, contains rich growth factors, effectively maintains the activity of cells, can obviously improve the survival rate of MenSCs in vitro, is stored for 7 days at 4 ℃, has the cell survival rate of over 90%, does not need to be recovered in use, is convenient to store, transport and clinically popularize, has high safety and good biocompatibility, can improve the retention time of MenSCs in uterus, and has an obvious treatment effect on female IUA diseases.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A polypeptide hydrogel for loading stem cells, the polypeptide hydrogel comprising: polypeptide A, polypeptide B, platelet rich plasma and placental amniotic membrane extract;
the polypeptide A comprises a self-assembly polypeptide, and the primary structural formula of the self-assembly polypeptide comprises a formula (1) or a formula (2);
formula (1): ac- (Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala)n-NH2Wherein n is an integer of 2 to 8;
formula (2): ac- (Lys-Ala-Glu-Ala-Lys-Ala-Glu-Ala)n-NH2Wherein n is an integer of 2 to 8;
the primary structural formula of the polypeptide B comprises a formula (3);
formula (3): arg-Gly-Asp.
2. The polypeptide hydrogel for loading stem cells according to claim 1, wherein the concentration of the polypeptide A in the polypeptide hydrogel is 0.28-1.60 mg/mL;
preferably, the concentration of the polypeptide B in the polypeptide hydrogel is 0.07-0.4 mg/mL;
preferably, the volume percentage of the platelet rich plasma in the polypeptide hydrogel is 2-10%, and the platelet rich plasma contains 1 × 109/L～30×109Platelets at a/L concentration;
preferably, the concentration of the placental amniotic membrane extract in the polypeptide hydrogel is 2.5-4 mg/mL.
3. The method for preparing a polypeptide hydrogel for stem cell loading according to claim 1 or 2, wherein the method comprises:
and mixing the solution of the polypeptide A and the solution of the polypeptide B to obtain a mixed solution, and mixing the mixed solution with the platelet-rich plasma and the placental amniotic membrane extract to obtain the polypeptide hydrogel.
4. Use of the polypeptide hydrogel for loading stem cells according to claim 1 or 2 for cell preservation;
preferably, the cells include any one of or a combination of at least two of mesenchymal stem cells of blood origin, mesenchymal stem cells of bone marrow, mesenchymal stem cells of umbilical cord, embryonic stem cells, mesenchymal stem cells of placenta, endometrial stem cells or induced pluripotent stem cells.
5. Use of the polypeptide hydrogel for loading stem cells of claim 1 or 2 for the preparation of a cell preparation;
preferably, the cells include any one of or a combination of at least two of mesenchymal stem cells of blood origin, mesenchymal stem cells of bone marrow, mesenchymal stem cells of umbilical cord, embryonic stem cells, mesenchymal stem cells of placenta, endometrial stem cells or induced pluripotent stem cells.
6. A cell preparation comprising the polypeptide hydrogel for stem cell loading of claim 1 or 2 and living cells;
preferably, the living cells include any one of or a combination of at least two of mesenchymal stem cells of blood origin, mesenchymal stem cells of bone marrow, mesenchymal stem cells of umbilical cord, embryonic stem cells, mesenchymal stem cells of placenta, endometrial stem cells or induced pluripotent stem cells;
preferably, the cell preparation further comprises any one or a combination of at least two of a preservative, an antioxidant and an antimicrobial;
preferably, the antioxidant comprises any one of or a combination of at least two of beneficial polyphenol, tea polyphenol, vitamin C or vitamin E;
preferably, the antibacterial agent comprises any one of carbomer, chitosan or nano silver or a combination of at least two of carbomer, chitosan or nano silver.
7. The cell preparation of claim 6, wherein the number of viable cells in the preparation is 1 x 10 and the number of viable cells is from P2 to P66～5×106And (4) respectively.
8. A method of preparing a cell preparation according to claim 6 or 7, said method comprising:
mixing the solution of the polypeptide A and the solution of the polypeptide B to obtain gel mother liquor, diluting the gel mother liquor to obtain gel working solution, mixing platelet-rich plasma, an antioxidant, an antibacterial agent and living cells to obtain cell mixed solution, and mixing the gel working solution, the cell mixed solution and the placenta amnion extract to obtain the cell preparation.
9. Use of the polypeptide hydrogel of claim 1 or 2 or the cell preparation of claim 6 or 7 for the preparation of a stem cell therapeutic.
10. A stem cell therapeutic agent comprising the cell preparation of claim 6 or 7;
preferably, the stem cell therapy medicament further comprises an adjuvant;
preferably, the adjuvant comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, a diluent, a preservative, an antioxidant, an antibacterial agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant or a pH regulator.
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