Large-scale preparation method of mesenchymal stem cell factors

A technology for large-scale preparation of mesenchymal stem cells, which is applied in the field of large-scale preparation of mesenchymal stem cell factors, can solve the problems of small amount of mesenchymal stem cell factors, difficulty in ensuring product uniformity, and unscalable separation and purification process, and achieve the goal of preparing The process conditions are stable and reasonable, and the product quality stability is realized, which is conducive to the effect of stability

Active Publication Date: 2017-05-31
北京银丰鼎诚生物工程技术有限公司
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Problems solved by technology

[0004] The current report on the preparation method of mesenchymal stem cell factors, such as the human-derived mesenchymal stem cell factor and its preparation method disclosed in Chinese patent application CN105543313A, collects and merges P1-P5 generation cells when they grow to a confluence of 75-85% The supernatant of the supernatant, and the supernatant is filtered and ultrafiltered to obtain the cytokine concentrate. This method fails to provide an effective induction medium, and the process of inducing cytokine secretion is not efficient enough. The number of mesenchymal stem cell factors obtained less, and this method needs to collect multiple generations of cell culture medium, the efficiency is low, the process is discontinuous, and it is easy to cause unnecessary contamination of the sample, and the method of multi-generation collection to prepare cytokines is difficult to ensure product uniformity. The purification process is not scalable, and it is difficult to adapt to mass production

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Embodiment 1

[0043] A large-scale preparation method of mesenchymal stem cell factors, comprising the following steps:

[0044] 1. Select mesenchymal stem cells, after in vitro primary culture and subculture to 4th generation, digest and count the 4th generation cells, and transfer them to the cell factory for expansion culture;

[0045] 2. When the expanded and cultured cells grow to a confluence of 70-85%, discard the original medium, add the induction medium to the cell factory, continue the culture, and collect the induction medium;

[0046] 3. Circulate and filter the collected induction medium through the hollow fiber membrane for 5 times, collect the first crude pure liquid, pump the first crude pure liquid to the tangential flow ultrafiltration membrane bag, and circulate and filter to the first crude pure liquid Concentrate the volume to 1 / 50 of the original volume, then add PBS buffer to the concentrated crude pure solution to make up to the original volume to obtain the second c...

Embodiment 2

[0049] A large-scale preparation method of mesenchymal stem cell factors, comprising the following steps:

[0050] 1. Select mesenchymal stem cells, after in vitro primary culture and subculture to 4th generation, digest and count the 4th generation cells, and transfer them to the cell factory for expansion culture;

[0051] 2. When the expanded and cultured cells grow to a confluence of 70-85%, discard the original medium, add the induction medium to the cell factory, continue the culture, and collect the induction medium;

[0052] 3. Circulate and filter the collected induction medium through the hollow fiber filter membrane for 8 times, collect the first crude pure liquid, pump the first crude pure liquid to the tangential flow ultrafiltration membrane bag, and circulate and filter to the first crude pure liquid Concentrate the volume to 1 / 30 of the original volume, then add PBS buffer to the concentrated crude pure solution to make up to the original volume to obtain the s...

Embodiment 3

[0055] A large-scale preparation method of mesenchymal stem cell factors, comprising the following steps:

[0056] 1. Select mesenchymal stem cells, after in vitro primary culture and subculture to 4th generation, digest and count the 4th generation cells, and transfer them to the cell factory for expansion culture;

[0057] 2. When the cells grow to a confluence of 70-85%, discard the original medium, and add induction medium according to half the volume of the original medium, and continue to cultivate and adjust the parameters: 37 ° C, 5% CO 2 , 5% O 2 After 24 hours, adjust the culture parameters to: 37°C, 5% CO 2 , 20%O 2 After 48 hours, half the amount of induction medium was collected, and an equal amount of fresh induction medium was added to the cell factory, and the parameters were adjusted to 37°C, 5% CO 2 , 5% O 2 , continue to cultivate; collect all induction medium after 72h;

[0058] 3. Pass the collected induction medium through a 0.1um hollow fiber filter...

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Abstract

The invention relates to a large-scale preparation method of mesenchymal stem cell factors. The large-scale preparation method comprises three steps of invitro culture of mesenchymal stem cells, induction and secretion of the mesenchymal stem cell factors and separation and purification of the mesenchymal stem cell factors, wherein an induction medium required by the step of the induction and secretion of the mesenchymal stem cell factors is prepared from a mixed medium of a DMEM (Dulbecco's Modified Eagle Medium), a PBS buffer solution and an L-alanyl-L-glutamine aqueous solution of which the volume ratio is (30 to 80):(20 to 60):(0.2 to 1.5) and an inducer. According to the large-scale preparation method of the mesenchymal stem cell factors, a single sample source is adopted to culture on a large scale, so that the uniformity of batch products is guaranteed, and the induction medium is adopted in the step of the induction and secretion of the mesenchymal stem cell factors, so that a large quantity of cell factors are stimulated to secrete, a formula is reasonable and effective, the conditions of the whole preparation process are stable and reasonable, and the large-scale preparation method is suitable for large-scale production.

Description

technical field [0001] The invention belongs to the technical field of cytokines, in particular to a large-scale preparation method of mesenchymal stem cell factors. Background technique [0002] Mesenchymal stem cells are derived from the mesoderm and ectoderm in the early stages of development and belong to pluripotent stem cells. It has attracted increasing attention because of its multi-directional differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication. [0003] The mechanism of action of mesenchymal stem cells is not completely clear. It is generally believed that the main mechanism is as follows: 1. Paracrine secretion of cell trophic factors, cell growth factors, anti-apoptotic factors, etc. 2. Replace or repair dead or damaged cells. 3. Direct contact with cells to regulate cell functions. At present, more and more studies have shown that the paracrine effect of mesenchymal stem cells plays ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/0797C07K1/14C12M3/00C12M1/12C12M1/00
CPCC07K1/14C12M37/02C12M47/10C12N5/0602C12N5/0623C12N2500/32C12N2500/34C12N2500/38C12N2500/90
Inventor 雷云霆董白翔
Owner 北京银丰鼎诚生物工程技术有限公司
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