CN114075534A

CN114075534A - Porous collagen microsphere carrier for stem cell 3D culture and preparation method thereof

Info

Publication number: CN114075534A
Application number: CN202010803363.4A
Authority: CN
Inventors: 华子昂; 刘宝全; 朱美瑛; 万君兴; 竹添; 张建; 赵凯龙
Original assignee: Individual
Current assignee: Wanshi Biotechnology Beijing Co ltd
Priority date: 2020-08-11
Filing date: 2020-08-11
Publication date: 2022-02-22

Abstract

Mixing collagen (or collagen degradation products) and calcium carbonate, carrying out superfine grinding, granulating to obtain solid particles, fixing the solid particles in high-concentration glutaraldehyde, removing the calcium carbonate by dilute acid solution containing glutaraldehyde under the stirring condition, dissolving to form a pore channel, simultaneously completing glutaraldehyde fixation of collagen in the microsphere carrier, cleaning the obtained microporous material to remove glutaraldehyde and calcium salt, drying at low temperature and carrying out aseptic treatment to obtain the porous collagen microsphere carrier for stem cell 3D culture, wherein the porous collagen microsphere carrier is used for stem cell 3D culture.

Description

Porous collagen microsphere carrier for stem cell 3D culture and preparation method thereof

Technical Field

The invention relates to the field of stem cell culture, in particular to a porous collagen microsphere carrier for stem cell 3D culture and a preparation method thereof.

Background

The stem cells have a variety of types and are widely applied in the fields of disease treatment, medical and beauty treatment, anti-aging, drug screening and the like. Stem cell culture has become a bottleneck for the development of the whole stem cell industry chain, and stem cell culture devices have become hot spots for the development of the stem cell culture field. According to the characteristics of stem cell culture devices, two categories of 2D culture and 3D culture are divided. The 2D culture is mainly carried out by enlarging a dish, and liquid is changed in batches and gas is supplied by a diffusion method. The 3D culture is mainly completed in a three-dimensional container, the composition of a culture solution can be regulated and controlled automatically, air can be supplied to the culture solution actively, the regulation and control means of the culture process are richer, and the 3D culture technology becomes the mainstream of the stem cell industry chain gradually.

The stem cell 3D culture carrier is a key element influencing a stem cell 3D culture industrial chain, and a plurality of manufacturers research and develop the stem cell 3D culture carrier, but most manufacturers adopt an organic solvent including benzene as a pore-forming agent when manufacturing the stem cell 3D culture carrier, particularly a porous microcarrier, so that the stem cell 3D culture carrier is potentially harmful to the cultured stem cells, and the research of the pore-forming agent friendly to the stem cells is urgently needed.

Meanwhile, the traditional stem cell culture adopts pancreatin to digest the stem cells, so that the stem cells are dissociated from an insoluble carrier; the stem cell separation method has great damage to stem cells, and the stem cell industry urgently needs stem cell culture carriers capable of being cracked mildly, so that the damage to the stem cells is reduced to the maximum extent in the cracking process of the carriers. A novel stem cell 3D culture carrier which can be cracked under the condition of not damaging stem cells, is porous, simulates the stem cell nest-generating design and is friendly to the stem cells has the development bottleneck of the stem cell industry.

Disclosure of Invention

Aiming at the defects, the invention provides a porous collagen microsphere carrier for stem cell 3D culture and a preparation method thereof, which substantially take a calcium carbonate material friendly to stem cells as a pore-forming agent, dissolve and remove calcium carbonate by using dilute acid and form internal pore channels of the carrier, and an organic solvent is not used in the whole process of manufacturing the carrier for stem cell 3D culture, so that the problem of organic solvent residue in the carrier for stem cell 3D culture caused by removing the organic solvent for pore-forming by using a volatilization method is effectively avoided. Meanwhile, the prepared porous collagen microsphere carrier for stem cell 3D culture can be mildly cracked by collagenase, and the damage to stem cells when the porous carrier is cracked and releases the stem cells is effectively avoided.

The invention overcomes the defects of the existing stem cell culture carrier, and the porous collagen microsphere carrier for stem cell 3D culture and the preparation method thereof provided by the invention have the following specific steps:

s1 mixing collagen (or collagen degradation product) and calcium carbonate in proportion, and micronizing;

s2, obtaining solid particles by using a solid granulation technology;

s3, after the solid particles are fixed in high-concentration glutaraldehyde, dissolving calcium carbonate in dilute acid solution containing glutaraldehyde to form a pore channel, and simultaneously fixing collagen in the microspheres by using glutaraldehyde;

s4, fully washing the porous microspheres which are fixed by collagen and dissolved by calcium carbonate, drying and dehydrating at low temperature, and detecting and storing after aseptic processing.

Preferably, collagen (or collagen degradation product) and calcium carbonate are mixed in proportion and then subjected to superfine grinding, and calcium carbonate is used as a grinding aid to ensure that the collagen (or collagen degradation product) is sufficiently ground and simultaneously promote the sufficient mixing of the collagen and the calcium carbonate.

Preferably, the mixing ratio of collagen (or collagen degradation product) to calcium carbonate is in the range of 1: 0.1-2.

Preferably, solid particles are obtained by using a solid granulation technique, the particle size being 0.1-1 mm.

Preferably, the solid particles are placed in a dilute acid solution containing glutaraldehyde, calcium carbonate which plays a role of occupying space in the particle forming process forms a pore channel after the dilute acid is dissolved to form a space for future cell growth, and meanwhile, glutaraldehyde enters a channel inside the particles to complete the fixation of the whole particles.

Preferably, the concentration of glutaraldehyde ranges from 0.1% to 5%.

Preferably, the dilute acid has a pH in the range of pH 3 to pH 7.

Preferably, the microspheres subjected to collagen fixation and calcium carbonate dissolution are thoroughly washed, glutaraldehyde and calcium salt are removed, and the microspheres are washed to neutrality and then dried at low temperature.

Preferably, the sterile treatment of the stem cell 3D culture carrier is accomplished using irradiation techniques.

Preferably, the collagen carrier for stem cell 3D culture not only satisfies the 3D culture preparation of MSC stem cells, but also is suitable for the 3D culture preparation of HSC stem cells.

Has the advantages that:

the invention provides a porous collagen microsphere carrier for stem cell 3D culture and a preparation method thereof, which substantially take a calcium carbonate material friendly to stem cells as a pore-forming agent, dissolve and remove calcium carbonate by using dilute acid, do not use an organic solvent in the whole process of manufacturing the collagen carrier for stem cell 3D culture, and effectively avoid the problem of organic solvent residue in the stem cell 3D culture carrier caused by removing the organic solvent for pore-forming by using a volatilization method. Meanwhile, the porous collagen microsphere carrier can be cracked by collagenase mildly, so that the damage to the prepared 3D culture stem cells during the cracking and releasing of the porous carrier is effectively avoided.

The present invention will be further described with reference to the following examples. The invention relates to related technologies, methods and equipment which have been patented by development teams, and only the main application of the related technologies, methods or equipment in the stem cell 3D culture preparation process is mainly illustrated in the embodiments, and the specific details of the patented technologies, methods or equipment are not described in detail.

Example 1

(1) Preparation of porous collagen microsphere carrier for stem cell 3D culture

S1 mixing medical gelatin and calcium carbonate according to the weight ratio of 1: carrying out superfine grinding at a mass mixing ratio of 0.8;

s2 solid particles with a particle size of about 0.3mm are obtained by an extrusion granulation technology. (ii) a

S3, fixing solid particles by using a 10% glutaraldehyde solution, then placing the fixed solid particles in a dilute acid solution containing glutaraldehyde, wherein the concentration range of the glutaraldehyde is 0.25%, the pH value range of the dilute acid is 4.5, performing collagen fixation by using the glutaraldehyde to keep the microsphere shape, dissolving calcium carbonate by using the dilute acid to form a pore channel, and forming the collagen carrier microsphere for stem cell 3D culture with multiple pore channels inside;

s4, fully washing the porous microspheres which are fixed by collagen and dissolved by calcium carbonate with ultrapure water, removing glutaraldehyde and calcium salt, washing to neutrality, and drying at low temperature. And (3) performing irradiation sterile treatment on the dried porous microspheres, and detecting and storing products after the sterile treatment.

(2) MSC stem cell 3D culture by using collagen carrier for stem cell 3D culture

S5, carrying out stem cell 3D culture proliferation by using a stem cell 3D culture instrument; the stem cell 3D culture instrument relates to the issued patents: 201910408085.X a three-dimensional simulation culture system of stem cells, 201910403665.X an adjustment method of gas balance in a stem cell culture container, and 201910408089.8 a 3D stem cell simulation culture device. The specific MSC stem cell 3D culture solution composition and preparation (see the reported patent; 202010763841.3 a proliferation culture medium for stem cell 3D simulation culture; 202010763837.7 a preparation method of the proliferation culture medium for stem cell 3D simulation culture):

s6 mixing the collagen carrier for stem cell 3D culture with MSC stem cell seeds, inoculating each million of MSCs with 100mg of microcarrier, and performing MSC stem cell 3D culture proliferation after inoculation.

S7 corresponding detection and preservation are carried out on the obtained MSC stem cells. And (3) detecting the surface antigen of the obtained MSC by a flow cytometer according to a standard operation method, wherein the detection result is as follows: CD29(99.87%), CD 44(99.98%), CD73 (99.89%), CD90 (99.68%), CD 105(99.56%); CD14 (0.13%), CD19 (0.11%), CD31 (0.18%), CD34 (0.07%), CD45 (0.09%), HLA-DR (0%); the functional index of the MSC stem cells exceeds the requirement of MSC surface antigen in China and abroad.

Example 2

(1) Preparation of collagen vector for 3D culture of stem cells

S1 mixing medical gelatin and calcium carbonate according to the weight ratio of 1: carrying out superfine grinding at a mass mixing ratio of 0.1;

s2 solid particles with a particle size of about 0.1mm are obtained by an extrusion granulation technology.

S3, placing solid particles in dilute acid solution containing glutaraldehyde, wherein the concentration range of the glutaraldehyde is 0.1%, the pH value range of the dilute acid is 6, utilizing the glutaraldehyde to fix collagen and keep the microsphere shape, utilizing the dilute acid to dissolve calcium carbonate to form a pore channel, and forming the collagen carrier microsphere for stem cell 3D culture with an internal microporous channel;

(2) MSC stem cell 3D culture by using collagen carrier for stem cell 3D culture

S7 corresponding detection and preservation are carried out on the obtained MSC stem cells. And (3) detecting the surface antigen of the obtained MSC by a flow cytometer according to a standard operation method, wherein the detection result is as follows: CD29(99.97%), CD 44(99.98%), CD73 (99.95%), CD90 (99.88%), CD 105(99.94%); CD14 (0.13%), CD19 (0.03%), CD31 (0.04%), CD34 (0.04%), CD45 (0.07%), HLA-DR (0%); the functional index of the MSC stem cells exceeds the requirement of MSC surface antigen in China and abroad.

Example 3

(1) Preparation of collagen vector for 3D culture of stem cells

S1 mixing collagen and calcium carbonate according to the ratio of 1: 2, carrying out superfine grinding at a mass mixing ratio;

s2 solid particles with a particle size of about 1mm are obtained by using an extrusion granulation technology.

S3, placing solid particles in dilute acid solution containing glutaraldehyde, wherein the concentration range of the glutaraldehyde is 5%, the pH value range of the dilute acid is 3, utilizing the glutaraldehyde to fix collagen and keep the microsphere shape, utilizing the dilute acid to dissolve calcium carbonate to form a pore channel, and forming the collagen carrier microsphere for stem cell 3D culture with an internal microporous channel;

(2) MSC stem cell 3D culture by using collagen carrier for stem cell 3D culture

S7 corresponding detection and preservation are carried out on the obtained MSC stem cells. And (3) detecting the surface antigen of the obtained MSC by a flow cytometer according to a standard operation method, wherein the detection result is as follows: CD29(99.87%), CD 44(99.88%), CD73 (99.88%), CD90 (99.97%), CD 105(99.74%), CD14 (0.15%), CD19 (0.05%), CD31 (0.09%), CD34 (0.04%), CD45 (0.05%), HLA-DR (0%); the functional index of the MSC stem cells exceeds the requirement of MSC surface antigen in China and abroad.

Example 4

(1) Preparation of collagen vector for 3D culture of stem cells

S1 mixing medical gelatin and calcium carbonate according to the weight ratio of 1: 1, carrying out superfine grinding at a mass mixing ratio;

s2 solid particles with a particle size of about 0.3mm are obtained by an extrusion granulation technology.

S3, placing solid particles in dilute acid solution containing glutaraldehyde, wherein the concentration range of the glutaraldehyde is 5%, the pH value range of the dilute acid is 4.5, utilizing the glutaraldehyde to fix collagen and keep the microsphere shape, utilizing the dilute acid to dissolve calcium carbonate to form a pore channel, and forming the collagen carrier microsphere for stem cell 3D culture with an internal microporous channel;

(2) MSC stem cell 3D culture by using collagen carrier for stem cell 3D culture

S7 corresponding detection and preservation are carried out on the obtained MSC stem cells. And (3) detecting the surface antigen of the obtained MSC by a flow cytometer according to a standard operation method, wherein the detection result is as follows: CD29(99.77%), CD 44(99.88%), CD73 (99.75%), CD90 (99.978%), CD 105(99.74%); CD14 (0.17%), CD19 (0.27%), CD31 (0.17%), CD34 (0.12%), CD45 (0.11%), HLA-DR (0%); the functional index of the MSC stem cells exceeds the requirement of MSC surface antigen in China and abroad.

Example 5

(1) Preparation of collagen vector for 3D culture of stem cells

s2 solid particles with a particle size of about 0.4mm are obtained by an extrusion granulation technology. (ii) a

S3, placing solid particles in dilute acid solution containing glutaraldehyde, wherein the concentration range of the glutaraldehyde is 0.2%, the pH value range of the dilute acid is 4.5, utilizing the glutaraldehyde to fix collagen and keep the microsphere shape, utilizing the dilute acid to dissolve calcium carbonate to form a pore channel, and forming the collagen carrier microsphere for stem cell 3D culture with an internal microporous channel;

(2) 3D culture of NK cells with collagen carrier for 3D culture of stem cells

s6 mixing the collagen carrier for stem cell 3D culture with NK cell seeds, inoculating the mixture according to the proportion of 200mg microcarrier for each million NK cells, and performing NK cell 3D culture proliferation after inoculation.

S7 corresponding detection and preservation of the obtained NK cells. The obtained NK cells are subjected to surface antigen detection of a flow cytometer according to a standard operation method, and the detection result is CD3⁺CD8⁺、CD3^-CD（56+16）⁺、CD3⁺CD56⁺Over 95 percent, the antigen representation meets the national and international standard requirements.

The porous collagen microsphere carrier for stem cell 3D culture and the preparation method thereof comprise any combination of the invention contents and the specific embodiment parts of the specification of the invention, are limited by space and do not describe all the schemes formed by the combination for the sake of conciseness of the specification. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. a porous collagen microsphere carrier for stem cell 3D culture and a preparation method thereof, is characterized in that, the steps of the method are as follows:

S1 Mix collagen (or collagen degradation product) with calcium carbonate in proportion and then superfinely pulverize;

S2 uses solid granulation technology to obtain solid particles;

S3, after the solid particles are fixed in high concentration glutaraldehyde, calcium carbonate is dissolved in a dilute acid solution containing glutaraldehyde to form pores, and at the same time, glutaraldehyde is used to fix the collagen inside the microspheres;

S4 Fully wash the porous microspheres with collagen fixation and calcium carbonate dissolution, dry and dehydrate at low temperature, and detect and store after aseptic processing.

2. A porous collagen microsphere carrier for stem cell 3D culture according to claim 1 and its preparation method, characterized in that S1 mixes collagen (or collagen degradation product) and calcium carbonate in proportion, and then performs ultrafine pulverization , Calcium carbonate is used as a grinding aid to ensure that the collagen (or collagen degradation product) is fully crushed, and at the same time, it promotes the full mixing of the two.

3. The mass mixing ratio of collagen (or collagen degradation product) according to claim 2 and calcium carbonate is in the range of 1:0.1-2.

4 . The porous collagen microsphere carrier for stem cell 3D culture according to claim 1 and the preparation method thereof, characterized in that S2 uses solid granulation technology to obtain solid particles with a particle size of 0.1-1 mm.

5. A kind of porous collagen microsphere carrier for stem cell 3D culture according to claim 1 and its preparation method, it is characterized in that, S3 is to place solid particles in a dilute acid solution containing glutaraldehyde, and during the particle formation process, the The occupied calcium carbonate forms pores after the dilute acid is dissolved to form the space for future cell growth, and it is also the channel for glutaraldehyde to enter the interior of the particle to complete the fixation of the entire particle. In the dilute acid solution of glutaraldehyde, the The concentration range is 0.1%-5%, and the pH range of dilute acid is pH 3-pH7.

6. The porous collagen microsphere carrier for stem cell 3D culture according to claim 1 and a preparation method thereof, wherein the post-processing of the porous collagen microsphere carrier comprises: fully washing and removing glutaraldehyde and dilute acid, Drying at low temperature ensures the physiological activity of collagen, and the method of sterilization by irradiation is preferred for aseptic treatment.

7. A porous collagen microsphere carrier for 3D culture of stem cells according to claim 1 and a preparation method thereof, wherein the porous collagen microsphere carrier after the 3D culture of stem cells can be specifically cleaved with collagenase, without damage to the stem cells cultured therein.

8 . The porous collagen microsphere carrier for stem cell 3D culture according to claim 1 and a preparation method thereof, characterized in that it not only meets the requirements for 3D culture preparation of MSC stem cells, but also is suitable for 3D culture preparation of HSC stem cells. 9 .

9. The application of the collagen carrier for 3D culture of stem cells prepared by the method in the production chain of stem cells.