CN112342192B - Continuous tangential flow stem cell separation and purification method - Google Patents
Continuous tangential flow stem cell separation and purification method Download PDFInfo
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Abstract
The invention provides a continuous tangential flow stem cell separation and purification method, which mainly comprises the following steps: s1: placing mesenchymal stem cells into a reactor, adding a microcarrier and a serum-free complete culture medium for culturing for 7 days, cleaning with normal saline, adding pancreatin for digestion for 30min, backfilling the serum-free complete culture medium to stop digestion, and preparing a pretreatment solution; s2: placing the pretreatment liquid in a liquid feeding bottle, connecting the liquid feeding bottle to a TFF system, connecting a permeate collecting bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline to the liquid feeding bottle, adding an equal amount of washing liquid into the liquid feeding bottle when the permeate collecting bottle collects a certain volume of waste liquid, emptying the permeate collecting bottle after the washing liquid is added, and stopping the system to obtain mesenchymal stem cells when the feed liquid in the liquid feeding bottle is left in a predicted volume; the cell recovery rate obtained by the continuous tangential flow stem cell separation and purification method provided by the invention is more than 80%, and the cell survival rate can reach more than 85%.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to a continuous tangential flow stem cell separation and purification method.
Background
Mesenchymal Stem cells (Mesenchymal Stem cells) are adult Stem cells with self-replicating renewal capacity and multidirectional differentiation potential, and can be differentiated into various histiocytes such as fat, cartilage, bone and the like under in vitro specific induction conditions. Its functions include immunosuppression, promoting the repair of body tissues and organs, promoting angiogenesis/regeneration, and supporting the proliferation and differentiation of hemopoietic stem cells. The umbilical cord derived mesenchymal stem cells are convenient to obtain materials, have no moral and ethical disputes, have a large number of cells and are fast in proliferation, and easy to passage, amplification and culture. In order to meet clinical requirements, mesenchymal stem cells need to be cultured in vitro in large scale. The collection of cells after culture is also an important part of obtaining stem cells for therapeutic use. The traditional centrifugation method needs manual operation and large-scale centrifugation equipment for collecting cells, and has the disadvantages of complicated process, long time consumption and pollution risk.
Disclosure of Invention
In order to solve the technical problems, the invention provides a continuous tangential flow stem cell separation and purification method.
The specific technical scheme of the invention is as follows:
the invention provides a continuous tangential flow stem cell separation and purification method, which mainly comprises the following steps:
s1: placing the mesenchymal stem cells into a reactor, adding a microcarrier and a serum-free complete culture medium for culture, cleaning with normal saline, adding pancreatin for digestion, backfilling the serum-free complete culture medium to stop digestion, and preparing a pretreatment solution;
s2: placing the pretreatment liquid in a liquid feeding bottle, connecting the liquid feeding bottle to a TFF system, connecting a permeate collecting bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline to the liquid feeding bottle, adding an equal amount of washing liquid into the liquid feeding bottle when the permeate collecting bottle collects a certain volume of waste liquid, emptying the permeate collecting bottle after the washing liquid is added, and stopping the system to obtain mesenchymal stem cells when the feed liquid in the liquid feeding bottle is left in a predicted volume;
wherein the incubation time with the addition of microcarriers and serum-free complete medium is preferably 7 days and the time for pancreatin digestion is preferably 30min, but other incubation times or digestion times are within the scope of the invention, and when a volume of waste stream from 1/4-1/2 pretreatment solution is collected in the permeate collection bottle, an equivalent volume of wash solution is added, the expected volume being 1/5-1/15 of the volume of waste stream emptied from the permeate collection bottle.
The method disclosed by the invention is used for separating and purifying the mesenchymal stem cells, the recovery rate of the cells can reach 80%, and the cell survival rate can reach 85%. And collecting the concentrated mesenchymal stem cells, adding a fresh culture medium for continuous culture, and observing the growth state of the cells on the 2 nd day. The cells adhere well and are in a slender fiber shape. The concentration, washing and filtering process has no influence on the activity and growth and proliferation capacity of cells, the microcarrier is honeycomb-shaped, the diameter is 0.5-10mm, the pore diameter of the microcapsule is 10-10000 nm, and the preparation method of the microcarrier comprises the following steps:
the preparation method of the microcarrier comprises the following steps:
(1) preparing balls: dripping 5L of 1% nanocrystallized sodium alginate solution into 0.5% calcium chloride solution to react to generate calcium alginate gel beads, wherein the volume ratio of the nanocrystallized sodium alginate solution to the calcium chloride solution is 1: 3;
(2) washing: removing redundant calcium chloride solution, washing with sterile purified water once, wherein the washing method comprises adding purified water, stirring at 50rpm for 5min, discarding washing solution to remove redundant liquid, and the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(3) gelatin coating: uniformly mixing 10L of 0.05% gelatin solution and 10mL of 0.3% glutaraldehyde solution to obtain a mixed solution, soaking calcium alginate micro-gel beads in the mixed solution, and reacting for 30min at the temperature of 20 ℃ and the stirring speed of 50rpm, wherein the volume ratio of the mixed solution to the nano sodium alginate solution is 1: 1;
(4) washing: after the coating reaction is finished, removing redundant mixed liquid, washing the mixture for three times by using sterile purified water, adding purified water, stirring the mixture at the rotating speed of 50rpm for 5min, and removing the redundant liquid by using the washing liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(5) neutralizing: adding 0.5% glycine solution, neutralizing for 2h at 40 deg.C and stirring speed of 50rpm, wherein the volume of glycine solution is the same as that of the mixed solution in step (3);
(6) washing: discarding the waste liquid after the reaction, washing the waste liquid for three times by using sterile purified water, adding the purified water, stirring the mixture at a rotating speed of 50rpm for 5min, discarding the washing liquid to remove redundant liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(7) coupling DEAE-HCl: adding 1mol/L NaOH solution into the washed calcium alginate micro-gel beads, stirring, and adding 0.5mol/L DEAE-HCl solution, and stirring; the ratio of the volume of the calcium alginate micro-gel beads to the volume of the NaOH solution and the volume of the DEAE-HCl solution is 1:1: 1; the specific conditions of the two times of stirring are as follows: the stirring speed is 50rpm, the stirring temperature is 60 ℃, and the stirring time is 0.5 h;
(8) washing: removing excess NaOH solution and DEAE-HCl solution, sequentially using sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free 2+ And Mg 2+ Washing with PBS buffer solution (5 min) at 50rpm, discharging the washing solution, and washing with sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free solution 2+ 、Mg 2+ The volume ratio of the PBS buffer solution to the calcium alginate micro-gel beads is 1:1, and a wet microcarrier is prepared;
(9) freeze-drying: freeze-drying the microcarrier obtained in the step (8), and performing radiation sterilization by using cobalt 60 gamma rays, wherein the radiation dose is 20KGy, so as to obtain the microcarrier.
Further, adding a serum-free complete culture medium into the bioreactor, and inoculating the mesenchymal stem cells and the microcarriers into the bioreactor together, wherein the density of the mesenchymal stem cells in the serum-free complete culture medium is 0.5-1x10 10 The density of the microcarrier in a serum-free complete culture medium is 4-6g/L, the microcarrier is cultured for 7 days, the temperature is controlled to be 37 ℃, the concentration of CO2 is 5%, the rotating speed of a bioreactor is 4-6rpm, the serum-free complete culture medium is respectively supplemented in days 2, 4 and 6, the volume ratio of the supplemented volume to the first serum-free complete culture medium is 1-3:3, 1-3:3 and 2-4:3, the culture solution is extracted into a liquid storage bottle in day 8, the microcarrier is cleaned by physiological saline for 5-20 minutes, the volume of the physiological saline is larger than the volume of the serum-free culture medium, pancreatin digestion cells are added after the physiological saline is removed, the temperature is set to be 37 ℃ during digestion, the rotating speed of the bioreactor is 4-6rpm, the digestion time is 20-40 minutes, the serum-free complete culture medium is refilled to terminate digestion, the ratio of the volume of the backfilled serum-free complete culture medium to the volume of the serum-free complete culture medium added for the first time is 1-3:2-4, and then the pretreatment solution is obtained.
Wherein, the step of washing the microcarrier with normal saline refers to the step of immersing the microcarrier with normal saline for 5-20 minutes, and pancreatin needs to be ensured to submerge all carriers during digestion.
Further, the washing solution is serum-free complete medium or normal saline.
Further, the washing solution in step S2 is serum-free complete medium or normal saline, and the washing time is 10-20 minutes.
Further, the serum-free complete medium comprises a basic medium and additives, wherein the basic medium is DMEM/F12 medium; the additive is human fibronectin with the concentration of 20-30 mug/ml, basic fibroblast growth factor with the concentration of 5-15ng/ml, human epidermal growth factor with the concentration of 5-15ng/ml, ITS with the concentration of 0.5-1.5% and human serum albumin with the concentration of 4-6%; NEAA with the concentration of 0.5 to 1.5 percent, hydrocortisone with the concentration of 0.05 to 0.15 mu mol/L and beta-mercaptoethanol with the concentration of 0.05 to 0.15 percent.
Further, the flow rate of the sample injection of the TFF system is 90-160 ml/min.
Further, the inlet end pressure of the TFF system is below 8 psi.
Further, the shear force of the TFF system is 1100-1800s -1 。
Further, the flux of the TFF system is 15-22L/h/m 2 。
Further, the pump speed of the TFF system was 110-.
Further, the filtration time of the TFF system is 15-20 min.
The cell recovery rate of the cells obtained by the continuous tangential flow stem cell separation and purification method provided by the invention is more than 80%, the cell survival rate can reach more than 85%, the concentrated mesenchymal stem cells are collected, a fresh culture medium is added for continuous culture, and the cell growth state is observed on day 2. The cells adhere well and are in a slender fiber shape. The concentration and washing process has no influence on the activity and growth and proliferation capacity of the cells.
Drawings
FIG. 1 is a microscopic image of cells purified by the method of example 1 after culturing in a medium.
Detailed Description
Example 1
Example 1 provides a continuous tangential flow stem cell isolation and purification method, which comprises the following steps:
s1: 2x10 10 Inoculating the mesenchymal stem cells and 15g of microcarrier into a bioreactor, adding a serum-free complete culture medium to 3L, culturing for 7 days, controlling the temperature to be 37 ℃, the concentration of CO2 to be 5 percent and the rotating speed of the bioreactor to be 5rpm, respectively supplementing 2L, 2L and 3L of serum-free complete culture medium on the days 2, 4 and 6, extracting the culture solution into a liquid storage bottle on the day 8, soaking and cleaning the microcarrier for 10 minutes by using physiological saline, removing the physiological saline, adding pancreatin digestive cells, setting the temperature to be 37 ℃ during digestion, setting the rotating speed of the bioreactor to be 5rpm, digesting for 30 minutes, backfilling the culture solution in the 2L liquid storage bottle, and stopping digestion to obtain a pretreatment solution;
s2: placing the pretreatment liquid in a material liquid bottle, connecting the material liquid bottle to a TFF system, connecting a permeate collection bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline to the material liquid bottle, adding 5L of serum-free complete culture medium into the material liquid bottle when the permeate collection bottle collects 5L of waste liquid, emptying the permeate collection bottle (namely emptying 5L of waste liquid) after the serum-free complete culture medium is completely added, and stopping the system when 500ml of feed liquid in the material liquid bottle is left, so as to obtain mesenchymal stem cells;
the preparation method of the microcarrier comprises the following steps:
(1) preparing balls: dripping 5L of 1% nanocrystallized sodium alginate solution into 0.5% calcium chloride solution to react to generate calcium alginate gel beads, wherein the volume ratio of the nanocrystallized sodium alginate solution to the calcium chloride solution is 1: 3;
(2) washing: removing redundant calcium chloride solution, washing with sterile purified water once, wherein the washing method comprises adding purified water, stirring at 50rpm for 5min, discarding washing solution to remove redundant liquid, and the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(3) gelatin coating: uniformly mixing 10L of 0.05% gelatin solution and 10mL of 0.3% glutaraldehyde solution to obtain a mixed solution, soaking calcium alginate micro-gel beads in the mixed solution, and reacting for 30min at the temperature of 20 ℃ and the stirring speed of 50rpm, wherein the volume ratio of the mixed solution to the nano sodium alginate solution is 1: 1;
(4) washing: after the coating reaction is finished, removing redundant mixed liquid, washing the mixture for three times by using sterile purified water, adding purified water, stirring the mixture at the rotating speed of 50rpm for 5min, and removing the redundant liquid by using the washing liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(5) neutralizing: adding 0.5% glycine solution, neutralizing for 2h at 40 deg.C and stirring speed of 50rpm, wherein the volume of glycine solution is the same as that of the mixed solution in step (3);
(6) washing: discarding the waste liquid after the reaction, washing the waste liquid for three times by using sterile purified water, adding the purified water, stirring the mixture at a rotating speed of 50rpm for 5min, discarding the washing liquid to remove redundant liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(7) coupling DEAE-HCl: adding 1mol/L NaOH solution into the washed calcium alginate micro-gel beads, stirring, and adding 0.5mol/L DEAE-HCl solution, and stirring; the ratio of the volume of the calcium alginate micro-gel beads to the volume of the NaOH solution and the volume of the DEAE-HCl solution is 1:1: 1; the specific conditions of the two times of stirring are as follows: the stirring speed is 50rpm, the stirring temperature is 60 ℃, and the stirring time is 0.5 h;
(8) washing: removing excess NaOH solution and DEAE-HCl solution, sequentially using sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free 2+ And Mg 2+ Washing with PBS buffer solution (5 min) at 50rpm, discharging the washing solution, and washing with sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free solution 2+ 、Mg 2+ The volume ratio of the PBS buffer solution to the calcium alginate bead is 1:1, preparing a wet microcarrier;
(9) freeze-drying: freeze-drying the microcarrier obtained in the step (8), and performing radiation sterilization by using cobalt 60 gamma rays, wherein the radiation dose is 20KGy, so as to prepare the microcarrier;
the serum-free complete culture medium comprises a basic culture medium and additives, wherein the basic culture medium is DMEM/F12 culture medium; the additive is human fibronectin with the concentration of 25 mug/ml, basic fibroblast growth factor with the concentration of 10ng/ml, human epidermal growth factor with the concentration of 10ng/ml, ITS with the concentration of 1 percent and human serum albumin with the concentration of 5 percent; NEAA with the concentration of 0.1 percent, hydrocortisone with the concentration of 0.1 mu mol/L and beta-mercaptoethanol with the concentration of 0.1 percent;
the parameters of the hollow fiber column of the TFF system are shown in Table 1, and the operating parameters of the TFF system are shown in Table 2.
Table 1 parameters of hollow fiber column.
TABLE 2 operating parameters of TFF System
| Flow rate of sample introduction | Speed of pump | Flux (W) | Transmembrane pressure | Shear force | Time of filtration |
| 110ml/min | 132rpm | 18L/h/m 2 | 0.3psi | 1300s -1 | 18min |
Example 2
Example 2 provides a continuous tangential flow stem cell separation and purification method, which comprises the following steps:
s1: 2x10 10 Inoculating the mesenchymal stem cells and 12g of microcarriers into a bioreactor, adding a serum-free complete culture medium to 3L, culturing for 7 days, controlling the temperature to be 37 ℃, the concentration of CO2 to be 5 percent and the rotating speed of the bioreactor to be 5rpm, respectively supplementing 2L, 2L and 3L of serum-free complete culture medium on the days 2, 4 and 6, extracting the culture solution into a liquid storage bottle on the day 8, cleaning the microcarriers for 10 minutes by using normal saline, removing the normal saline, adding pancreatin digestive cells, setting the temperature to be 37 ℃ during digestion, setting the rotating speed of the bioreactor to be 4rpm, digesting for 20 minutes, backfilling 2L of serum-free complete culture to terminate digestion, and obtaining a pretreatment solution;
s2: placing the pretreatment liquid in a material liquid bottle, connecting the material liquid bottle to a TFF system, connecting a permeate collection bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline to the material liquid bottle, adding 5L of serum-free complete culture medium into the material liquid bottle when the permeate collection bottle collects 5L of waste liquid, emptying the permeate collection bottle (namely emptying 5L of waste liquid) after the serum-free complete culture medium is completely added, and stopping the system when 1000ml of feed liquid in the material liquid bottle is left, so as to obtain mesenchymal stem cells;
the preparation method of the microcarrier is the same as that of the microcarrier in the embodiment 1, and the serum-free complete culture medium comprises a basal culture medium and an additive, wherein the basal culture medium is DMEM/F12 culture medium; the additive is human fibronectin with the concentration of 20 mu g/ml, basic fibroblast growth factor with the concentration of 5ng/ml, human epidermal growth factor with the concentration of 5ng/ml, ITS with the concentration of 0.5 percent and human serum albumin with the concentration of 4 percent; NEAA with the concentration of 0.5 percent, hydrocortisone with the concentration of 0.05 mu mol/L and beta-mercaptoethanol with the concentration of 0.05 percent;
the parameters of the hollow fiber column of the TFF system are shown in Table 1, and the operating parameters of the TFF system are shown in Table 3.
TABLE 3 operating parameters of TFF System
| Flow rate of sample introduction | Pump speed | Flux (W) | Transmembrane pressure | Shear force | Time of filtration |
| 90ml/min | 110rpm | 15L/h/m 2 | 0.2psi | 1100s -1 | 20min |
Example 3
Example 3 provides a continuous tangential flow stem cell isolation and purification method, which comprises the following steps:
s1: 2x10 10 Inoculating mesenchymal stem cells and 18g microcarrier into a bioreactor, adding serum-free complete culture medium to 3L, culturing for 7 days, controlling the temperature at 37 ℃, the concentration of CO2 at 5% and the rotation speed of the bioreactor at 5rpm, respectively supplementing 2L, 2L and 3L of serum-free complete culture medium on days 2, 4 and 6, extracting the culture solution to a liquid storage bottle on day 8, washing the microcarrier with physiological saline for 10 minutes, removing the physiological saltAdding pancreatin to digest cells after adding water, setting the temperature at 37 ℃ during digestion, setting the rotating speed of the bioreactor at 6rpm, setting the digestion time at 40 minutes, and backfilling the culture solution in a 2L liquid storage bottle to stop digestion to obtain a pretreatment solution;
s2: placing the pretreatment liquid in a material liquid bottle, connecting the material liquid bottle to a TFF system, connecting a permeate collection bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline to the material liquid bottle, adding 5L of serum-free complete culture medium into the material liquid bottle when the permeate collection bottle collects 5L of waste liquid, emptying the permeate collection bottle (namely emptying the 5L of waste liquid) after the serum-free complete culture medium is completely added, and stopping the system when the material liquid in the material liquid bottle is 250ml, thus obtaining mesenchymal stem cells;
the preparation method of the microcarrier is the same as that in the embodiment 1, and the serum-free complete culture medium comprises a basal culture medium and an additive, wherein the basal culture medium is DMEM/F12; the additive is human fibronectin with the concentration of 30 mu g/ml, basic fibroblast growth factor with the concentration of 15ng/ml, human epidermal growth factor with the concentration of 15ng/ml, ITS with the concentration of 1.5 percent and human serum albumin with the concentration of 6 percent; 1.5% NEAA, 0.15% micromole/L hydrocortisone, 0.15% beta-mercaptoethanol;
the parameters of the hollow fiber column of the TFF system are shown in Table 1, and the operating parameters of the TFF system are shown in Table 4.
TABLE 4 operating parameters of TFF System
| Flow rate of sample introduction | Speed of pump | Flux of a magnetic material | Transmembrane pressure | Shear force | Time of filtration |
| 160ml/min | 173rpm | 22L/h/m 2 | 0.5psi | 1800s -1 | 15min |
Comparative example 1
The present comparative example provides a continuous tangential flow stem cell separation and purification method, and the difference from example 1 is that the working parameters of the TFF system are shown in Table 5.
TABLE 5 operating parameters of TFF System
| Flow rate of sample introduction | Speed of pump | Flux (W) | Transmembrane pressure | Shear force | Time of filtration |
| 60ml/min | 70rpm | 10L/h/m 2 | 0psi | 650s -1 | 40min |
Comparative example 2
The present comparative example provides a continuous tangential flow stem cell separation and purification method, and the difference from example 1 is that the working parameters of the TFF system are shown in Table 6.
TABLE 6 operating parameters of TFF System
| Flow rate of sample introduction | Speed of pump | Flux (W) | Transmembrane pressure | Shear force | Time of filtration |
| 200ml/min | 220rpm | 31L/h/m 2 | 1.1psi | 2300s -1 | 10min |
Test example 1
Mesenchymal stem cells were isolated and purified by the methods provided in examples 1 to 3 and comparative examples 1 to 2 of the present invention, the number of cells harvested by each set of methods, the cell viability rate, and the cell recovery rate were calculated, and the number of cells before isolation after culturing in examples 1 to 3 and comparative examples 1 to 2 was 1.78 x10 11 The calculation formula of the cell recovery rate is as follows: cell count/1.78 × 10 11 The cell number is measured by counting under a microscope; the cell viability rate, which is (total number of cells-number of dead cells)/total number of cells, was calculated according to the following formula, and the test results are shown in table 7; the cells obtained in example 1 after 24 hours of culture were observed under a microscope, and the results are shown in FIG. 1 at a magnification of 100.
TABLE 7 cell viability test results by each set of methods.
As can be seen from Table 1, the continuous tangential flow stem cell separation and purification method provided by the invention can be used for recovering cells, the cell recovery rate is more than 80%, the cell viability can reach more than 85%, the concentrated mesenchymal stem cells are collected, fresh culture medium is added for continuous culture, and the growth state of the cells is observed on day 2. The cells adhere well, the shape is in a slender fiber shape, and the activity and the growth and proliferation capacity of the cells are not influenced in the process of concentration and washing.
Therefore, the invention is not limited to the specific embodiments and examples, but rather, all equivalent variations and modifications are within the scope of the invention as defined in the claims and the specification.
Claims (10)
1. A continuous tangential flow stem cell separation and purification method is characterized by mainly comprising the following steps:
s1: placing the mesenchymal stem cells into a reactor, adding a microcarrier and a serum-free complete culture medium for culture, cleaning with normal saline, adding pancreatin for digestion, backfilling the serum-free complete culture medium to stop digestion, and preparing a pretreatment solution;
s2: placing the pretreatment liquid in a liquid feeding bottle, connecting the liquid feeding bottle with a TFF system, connecting a permeate collecting bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline with the liquid feeding bottle, adding an equal amount of washing liquid into the liquid feeding bottle when the permeate collecting bottle collects a certain volume of waste liquid, emptying the permeate collecting bottle after the washing liquid is added, and stopping the system when the feed liquid in the liquid feeding bottle has a residual preset volume to obtain mesenchymal stem cells;
the preparation method of the microcarrier comprises the following steps:
(1) preparing balls: dripping 5L of 1% nanocrystallized sodium alginate solution into 0.5% calcium chloride solution to react to generate calcium alginate gel beads, wherein the volume ratio of the nanocrystallized sodium alginate solution to the calcium chloride solution is 1: 3;
(2) washing: removing redundant calcium chloride solution, washing with sterile purified water once, wherein the washing method comprises adding purified water, stirring at 50rpm for 5min, discarding washing solution to remove redundant liquid, and the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(3) gelatin coating: uniformly mixing 10L of 0.05% gelatin solution and 10mL of 0.3% glutaraldehyde solution to obtain a mixed solution, soaking calcium alginate micro-gel beads in the mixed solution, and reacting for 30min at the temperature of 20 ℃ and the stirring speed of 50rpm, wherein the volume ratio of the mixed solution to the nano sodium alginate solution is 1: 1;
(4) washing: after the coating reaction is finished, removing redundant mixed liquid, washing the mixture for three times by using sterile purified water, adding purified water, stirring the mixture at the rotating speed of 50rpm for 5min, and removing the redundant liquid by using the washing liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(5) neutralizing: adding 0.5% glycine solution, neutralizing for 2h at 40 deg.C and stirring speed of 50rpm, wherein the volume of glycine solution is the same as that of the mixed solution in step (3);
(6) washing: discarding the waste liquid after the reaction, washing the waste liquid for three times by using sterile purified water, adding the purified water, stirring the mixture at a rotating speed of 50rpm for 5min, discarding the washing liquid to remove redundant liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(7) coupling DEAE-HCl: adding 1mol/L NaOH solution into the washed calcium alginate micro-gel beads, stirring, and adding 0.5mol/L DEAE-HCl solution, and stirring; the ratio of the volume of the calcium alginate micro-gel beads to the volume of the NaOH solution to the volume of the DEAE-HCl solution is 1:1: 1; the specific conditions of the two times of stirring are as follows: the stirring speed is 50rpm, the stirring temperature is 60 ℃, and the stirring time is 0.5 h;
(8) washing: removing excess NaOH solution and DEAE-HCl solution, sequentially using sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free 2+ And Mg 2+ The PBS buffer solution of (1), stirring at 50rpm for 5min each time, discharging the washing solution, and washing with sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and no Ca 2+ 、Mg 2+ The volume ratio of the PBS buffer solution to the calcium alginate micro-gel beads is 1:1, and a wet microcarrier is prepared;
(9) freeze-drying: freeze-drying the microcarrier obtained in the step (8), and performing radiation sterilization by using cobalt 60 gamma rays, wherein the radiation dose is 20KGy, so as to obtain the microcarrier.
2. The continuous tangential flow stem cell separation and purification method of claim 1, wherein the pretreatment solution of step S1 is prepared by the following steps: adding a serum-free complete culture medium into a bioreactor, and then inoculating the MSC cells and the microcarriers into the bioreactor together, wherein the density of the mesenchymal stem cells in the serum-free complete culture medium is 0.5-1x10 10 The density of the microcarrier in a serum-free complete culture medium is 4-6g/L, the microcarrier is cultured for 7 days, the temperature is controlled to be 37 ℃, the concentration of CO2 is 5%, the rotating speed of a bioreactor is 4-6rpm, the serum-free complete culture medium is respectively supplemented in days 2, 4 and 6, the volume ratio of the supplemented volume to the first serum-free complete culture medium is 1-3:3, 1-3:3 and 2-4:3, the culture solution is extracted into a liquid storage bottle in day 8, the microcarrier is washed by physiological saline for 5-20 minutes, the volume of the physiological saline is larger than the volume of the serum-free culture medium, pancreatin digestive cells are added after the physiological saline is removed, the temperature is set to be 37 ℃ during digestion, and biological reverse reaction is setThe rotating speed of the reactor is 4-6rpm, the digestion time is 20-40 minutes, the serum-free complete culture medium is backfilled to terminate the digestion, and the volume ratio of the backfilled serum-free complete culture medium to the first-time added serum-free complete culture medium is 1-3:2-4, so that the pretreatment solution is obtained.
3. The method for separating and purifying continuous tangential flow stem cells of claim 1, wherein the washing solution in step S2 is serum-free complete culture medium or normal saline, and the washing time is 10-20 minutes.
4. The continuous tangential flow stem cell separation and purification method of claims 1-2, wherein the serum-free complete medium comprises a basal medium and additives, and the basal medium is DMEM/F12 medium; the additive is human fibronectin with the concentration of 20-30 mug/ml, basic fibroblast growth factor with the concentration of 5-15ng/ml, human epidermal growth factor with the concentration of 5-15ng/ml, ITS with the concentration of 0.5-1.5% and human serum albumin with the concentration of 4-6%; NEAA with the concentration of 0.5-1.5%, hydrocortisone with the concentration of 0.05-0.15 mu mol/L, and beta-mercaptoethanol with the concentration of 0.05-0.15%.
5. The continuous tangential flow stem cell separation and purification method of claim 1, wherein the sample injection flow rate of the TFF system is 90-160 ml/min.
6. The method for separating and purifying continuous tangential flow stem cell as claimed in claim 1, wherein the shear force of the TFF system is 1100-1800s -1 。
7. The method for the isolation and purification of continuous tangential flow stem cells according to claim 1, wherein the pressure at the inlet end of the TFF system is lower than 8 psi.
8. The method for separating and purifying the continuous tangential flow stem cells of claim 1, wherein the TFF system has a flux of 15-22L/m 2 ·h。
9. The method for separating and purifying continuous tangential flow stem cells of claim 1, wherein the pump speed of the TFF system is 110-.
10. The method for separating and purifying the continuous tangential flow stem cells of claim 1, wherein the filtration time of the TFF system is 15-20 min.
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