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Method for serum-free domestication and culture of human mesenchymal stem cells

A technique for culturing and domesticating stem cells, applied in the field of human mesenchymal stem cell culture, can solve the problems of unsatisfactory growth of stem cells and high cost, and achieve the effects of improving safety and reducing costs.

Pending Publication Date: 2018-10-12
刘馨
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the composition of serum-free medium is clear, the cost is high, and there are problems such as unsatisfactory growth of stem cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] ——The preparation method of human mesenchymal stem cell injection, the following steps are adopted:

[0015] Placenta-derived MSC cells mainly include mesenchymal stem cells derived from umbilical cord and amniotic membrane. The following steps are used for culturing: Treatment of umbilical cord and amniotic membrane: The umbilical cord includes a vein and two arteries, surrounded by Wharton's Jelly, and the outer layer Wrapped by amnion-derived epithelium, from a developmental point of view, the umbilical cord is the pathway through which stem cells are formed. Studies have shown that the connective tissue of the human umbilical cord is a rich source of mesenchymal stem cells. The treatment method is to first peel off the umbilical arterial vein and the outer epithelium, leaving the gelatinous tissue (Wharton's jelly), and resect the parts with pinch marks and congestion on both sides.

[0016] The amniotic membrane is the innermost layer of the fetal membrane, a thin ...

Embodiment 2

[0030] ——The preparation of the concentrated essence or freeze-dried powder of human mesenchymal stem cell secretion components adopts the following process steps:

[0031] The components secreted by human mesenchymal stem cells include microcapsules with a diameter greater than 200nm and exosomes with a diameter of 30-150nm. These vesicles have a lipid bilayer membrane structure.

[0032] MSC cells were cultured using the method of the first embodiment above. When the confluence of the cells reached 80%, the cells were washed with PBS, and serum-free medium was added, including but not limited to MesenGro hMSC medium (StemRD, San Francisco, CA, USA), and then the cells were incubated at 37°C, 5% CO2 for 48 hours, This conditioned culture is based on centrifugation at 500g for 10 minutes at 4°C, and the supernatant is centrifuged at 1000g for 10 minutes at 4°C.

[0033] The supernatant was filtered with 0.22-μm (Steritop™; Millipore), concentrated 5 to 10 times with an ultraf...

Embodiment 3

[0038] ——The preparation of human mesenchymal stem cell facial mask adopts the following process steps:

[0039] MSC cells were cultured using the method in Example 1 above.

[0040] When the confluence of the cells reached 80% and the culture time in serum-free medium reached 40 hours, the culture supernatant was harvested, and the culture supernatant was centrifuged at 1000g at 4°C for 5 minutes to remove cell debris. The supernatant was filtered with 0.22-μm (Steritop™; Millipore), concentrated 5 to 10 times with an ultrafiltration membrane with a molecular weight cut-off of 50KD, collected the retentate, and replaced the solvent with 20mM PBS or normal saline by ultrafiltration. Freeze and thaw three times at room temperature and -20°C, aliquot and store at -80°C. When using, add it to the mask base solvent in a certain proportion, such as but not limited to 1% hyaluronic acid + 90% water for injection + 10% glycerin, shake slightly to dissolve and mix well.

[0041] The...

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Abstract

The invention discloses a method for serum-free domestication and culture of human mesenchymal stem cells. According to the method, human placenta-derived mesenchymal stem cells are allowed to adapt to an in-vitro serum-free culture system, and cell density and cell viability similar to the cell density and cell viability of serum culture are obtained; and compared with conventional culture methods for human mesenchymal stem cells, the method provided by the invention is more economical and effective. With the method, the expression levels of bFGF, PDGF and VEGF associated with blood vessel growth are increased compared with undomesticated cells. The domesticated cells can be prepared for preparing stem cell preparations for intravenous injection; and active factor components secreted by the domesticated cells can be used as components of a mask, an eye mask and a hair mask.

Description

technical field [0001] The present invention relates to a culture method of human mesenchymal stem cells, and more specifically, the present invention relates to a serum-free in vitro culture of human mesenchymal stem cells by using a unique and safe acclimation culture method. Background technique [0002] Stem cells are the ancestors of human cells, and all cells in our body are derived from stem cells. When cells in the body age, die or become damaged and degenerate, stem cells grow and transform into cells that can replace them. As seed cells, it is mainly used clinically to treat various refractory diseases of tissue cells and organ damage that cannot be naturally repaired by the body; as immune regulatory cells, it is used to treat immune rejection and autoimmune diseases. Human mesenchymal stem cells (human mesenchymal stem cells, HMSC) is an important member of the stem cell family, derived from the mesoderm in the early development, belonging to pluripotent stem ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61K9/08A61P43/00A61P37/06A61P37/02A61P9/00A61K8/99A61Q19/00A61Q7/00A61Q19/02A61Q19/08
CPCA61Q7/00A61Q19/00A61Q19/005A61Q19/02A61Q19/08A61K8/99A61K9/0019A61K9/08A61K35/28C12N5/0668C12N2500/90
Inventor 刘馨马波
Owner 刘馨
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