Multi-batch primary separation method of same-donor-derived human mesenchymal stem cells

A separation method and stem cell technology, applied in the field of multi-batch primary separation, can solve problems such as high price, limited cell viability, and decreased mesenchymal stem cell viability, and achieve the effect of avoiding risks and drawbacks

Active Publication Date: 2018-07-06
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In fact, limited by factors such as the inherent culture area and enzyme digestion efficiency of the separation vessel, it is difficult to fully and completely separate the mesenchymal stem cells rich in umbilical cord tissue after only one treatment and culture
Therefore, according to the existing technical scheme, the utilization efficiency of umbilical cord samples collected from clinic and the isolation yield of hUC-MSCs are both low.
[0016] Second: It takes a long time to isolate hUC-MSCs (existing tissue block attachment method)
In summary, the time to obtain hUC-MSCs using the existing tissue block attachment method is generally about 20 days, and it is difficult to provide sufficient mesenchymal stem cells for relevant experimental research or clinical treatment in a short period of time.
[0017] Third: The isolated cells have limited viability and high separation costs (enzyme digestion method)
Reagents such as collagenase and trypsin are expensive, and enzymatic hydrolysis treatment can easily lead to a decline in the viability of mesenchymal stem cells

Method used

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  • Multi-batch primary separation method of same-donor-derived human mesenchymal stem cells
  • Multi-batch primary separation method of same-donor-derived human mesenchymal stem cells
  • Multi-batch primary separation method of same-donor-derived human mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1 A method for the isolation of multiple batches of primary human mesenchymal stem cells from the same donor

[0103] The method is as above, wherein, step (5) uses different concentrations of fetal bovine serum (FBS) or human AB serum to soak the small pieces of umbilical cord tissue in the previous step for 15 minutes, and obtain primary cells after plating.

[0104] Primary cells refer to the earliest algebraic cells directly derived from in vitro cultured tissues. In the present invention, the mesenchymal stem cells obtained after culturing or incubating the small tissue pieces and the original culture supernatant without passage are collectively referred to as primary cells.

[0105] For each group, the earliest appearance time of primary cells, the time of the first passage, the success rate of tissue block culture (the number of tissue blocks that successfully freed cells / the total number of inoculated tissue blocks × 100%), the number of primary cells ( ...

Embodiment 2

[0111] Example 2 A method for the isolation of multiple batches of primary human mesenchymal stem cells from the same donor

[0112] The method is as above, step (5) soak the umbilical cord tissue pieces in the previous step with 100% fetal bovine serum for 15 minutes. The primary isolation time and yield of each batch of hUC-MSCs were compared. Among them, group A is the first culture group of umbilical cord tissue blocks; group B-t is the mesenchymal stem cells (primary cells) obtained from the recovered umbilical cord tissue blocks after the first culture through the second culture; group B-s is the supernatant of the first culture of tissue blocks The mesenchymal stem cells (primary cells) obtained after incubation with B-t solution alone; the C-t group is the mesenchymal stem cells (primary cells) obtained from the umbilical cord tissue block (recovered from the B-t group) through the third culture; the C-s group is the tissue block Mesenchymal stem cells (primary cells)...

Embodiment 3

[0137] Example 3 A method for the isolation of multiple batches of primary human mesenchymal stem cells from the same donor

[0138] The method is as described above, in step (5), 100% human AB serum is used to soak the small pieces of umbilical cord tissue in the previous step for 15 minutes. The primary isolation time and yield of each batch of hUC-MSCs were compared. Among them, group A is the first culture group of umbilical cord tissue blocks; group B-t is the mesenchymal stem cells (primary cells) obtained from the recovered umbilical cord tissue blocks after the first culture through the second culture; group B-s is the supernatant of the first culture of tissue blocks The mesenchymal stem cells (primary cells) obtained after incubation with B-t solution alone; the C-t group is the mesenchymal stem cells (primary cells) obtained from the umbilical cord tissue block (recovered from the B-t group) through the third culture; the C-s group is the tissue block Mesenchymal s...

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Abstract

The invention discloses a multi-batch primary separation method of same-donor-derived human mesenchymal stem cells, and belongs to the field of cell culture. According to the method, a donor tissue istreated to obtain 1 to 5 mm <3> donor tissue small blocks, and the donor tissue small blocks are soaked with fetal bovine serum or human AB serum for processing, and then subjected to conventional tissue block adherent culture. While harvesting of the mesenchymal stem cells, the donor tissue small blocks are transferred to a new culture plate for continuously culturing, and the culture supernatant of the tissue blocks is recovered and continuously incubated to achieve high-quality, multi-batch stable separation of the same-donor-derived human mesenchymal stem cells. The morphology, proliferation and differentiation ability and immunophenotype of the cells obtained by method remain unchanged among batches. Compared with a traditional tissue block adherence method and an enzymatic digestionmethod, the method avoids the waste of clinical specimen resources, saves human and material resources of clinical links, and improves the separation efficiency and yield of the mesenchymal stem cells.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to a multi-batch primary separation method of human mesenchymal stem cells from the same donor source. Background technique [0002] Mesenchymal stem cells (MSCs) have a high degree of self-renewal and multi-directional differentiation capabilities, not only can differentiate into mesenchymal tissue cells such as fat, bone, cartilage, and muscle, but also have low immunogenicity and immunosuppressive effects. It is an important seed cell for the treatment of tissue engineering and cell replacement therapy, transplantation immunity and autoimmune diseases. [0003] Friedenstein and colleagues first grew mesenchymal stem cells from bone marrow in the 1970s. In recent years, researchers have successively isolated MSCs from other tissues, such as fat, deciduous teeth, and neonatal umbilical cord, placenta, amniotic membrane, and umbilical cord blood. Among them, the umbilical c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2509/00
Inventor 袁茵鲁欣匡梅娜黄思瑞
Owner GUANGDONG PHARMA UNIV
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