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Serum-free medium and preparation method thereof

A serum-free medium and basal medium technology, applied in biochemical equipment and methods, culture process, tissue culture and other directions, can solve the problems of unclear composition, affecting the standardized culture of mesenchymal stem cells, large quality differences, etc. Clear source of ingredients, good batch-to-batch reproducibility, and easy-to-obtain results

Active Publication Date: 2021-06-15
JIANGSU PURECELL BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of the problems existing in the prior art, for example, the composition of the mesenchymal stem cell culture medium based on serum or platelet lysate (PL) is unclear, and the quality varies greatly between different batches, which affects the standardized cultivation of mesenchymal stem cells. Defects

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  • Serum-free medium and preparation method thereof
  • Serum-free medium and preparation method thereof
  • Serum-free medium and preparation method thereof

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preparation example Construction

[0100] In the second aspect of the present invention, there is provided a method for preparing the serum-free medium of the first aspect, comprising the step of mixing the basal medium and the additive of the serum-free medium.

[0101] Further, the preparation method of the serum-free medium includes preparing the lysate of the basal medium, and preparing the lysate of the additive of the serum-free medium; then mixing the lysate of the basal medium with the lysate of the additive of the serum-free medium Homogenize and filter to obtain serum-free medium.

[0102] In some specific embodiments, the preparation method of the additive of serum-free medium comprises the following steps:

[0103] After mixing the dissolved solutions of the first component, the second component and the third component, filtering to obtain a mixed dissolved solution;

[0104]The mixed solution is pre-frozen, sublimated and dried to obtain the additive of freeze-dried powder.

[0105] Prepare mix...

Embodiment 1

[0123] The present embodiment provides a basal medium, and the preparation steps of the basal medium are as follows:

[0124] Dissolve the amino acid components in Table 2, the vitamin components in Table 3, the inorganic salt components in Table 4, the lipid components in Table 5, and other components in Table 6 in 1L of water for injection, and dissolve them fully Afterwards, mix well and filter to obtain the basal medium.

[0125] Basal medium: 17.4g; 1L

[0126] Table 2 Amino Acids

[0127] Reagent name Working concentration (mg / L) Reagent name Working concentration (mg / L) Glycine 26.25 Leucine 111.45 Alanine 13.35 Lysine 163.75 arginine 273.9 Methionine 32.34 Asparagine 20.7 Phenylalanine 68.48 Aspartic acid 19.95 proline 28.75 cysteine 17.56 serine 36.75 cystine 55.29 threonine 101.05 glutamic acid 22.05 Tryptophan 19.22 Glutamine 265 Tyrosine 91.79 Histidine 73.48 V...

Embodiment 2

[0137] The additives of the serum-free medium were prepared according to the components provided in Table 7, and the additives were specifically lyophilized powder. The preparation method of additive comprises the steps:

[0138] (1) Dissolve the first component, the second component and the third component in Table 7 respectively, mix the solution of the three types of components, mix well and filter

[0139] (2) Pre-freezing: the temperature in the box drops to -45°C and keeps for 3 hours

[0140] (3) Sublimation: lower the temperature of the condenser to below -55°C, and evacuate the entire system;

[0141] Control the degree of vacuum below 10pa, control the temperature of the separator at 25°C, and sublimate and dry for 24 hours.

[0142] (4) Drying: increase the temperature of the plate layer to 7°C and dry for 5 hours;

[0143] (5) After the freeze-drying is completed, input the mixed gas into the freeze-drying box to make the pressure in the box reach 1.1 standard a...

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Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a serum-free medium and a preparation method thereof. According to the serum-free medium disclosed by the invention, animal-derived components such as serum and platelet lysis buffer are replaced by an additive of serum free culture. The serum-free medium has the advantages of clear source of ingredients, good repeatability between batches and high clinical safety, can provide various substance components required by rapid growth and proliferation of mesenchymal stem cells, can keep the characteristics of the stem cells, can meet the requirements of in-vitro culture of mesenchymal stem cells from different sources such as fat, bone marrow, umbilical cord, umbilical cord blood and placenta.

Description

technical field [0001] The invention belongs to the technical field of cell culture, in particular, the invention relates to a serum-free medium and a preparation method thereof. Background technique [0002] Mesenchymal stem cells (MSCs) widely exist in various human tissues and organs such as adult bone marrow, fat, dental pulp, perinatal umbilical cord blood, umbilical cord, placenta, etc. Adult stem cells with increasing ability and immune regulation function, mesenchymal stem cells still have the characteristics of stem cells after multiple passages and expansion, and the surface antigen is not obvious, the rejection of allogeneic transplantation is relatively light, and the matching requirements are not strict. It is autologous and allogeneic cell transplantation. Ideal for seeding cells with treatments. [0003] Mesenchymal stem cells were first discovered in bone marrow, and can also be isolated from fat, umbilical cord blood, placenta, umbilical cord, amniotic flui...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0662C12N2500/90
Inventor 孙振华施坤宁王星
Owner JIANGSU PURECELL BIOMEDICAL TECH CO LTD
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