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Method for expanding hemopoietic stem cell under three-dimensional condition

A technology of hematopoietic stem cells and stem cells, which is applied to blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of lack of culture medium, large fluid shear force, stem/progenitor cell differentiation, etc., to improve the culture effect. , the effect of reducing shear force and avoiding immune rejection

Inactive Publication Date: 2006-09-27
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1) The lack of effective mixing of the culture medium leads to the formation of a concentration gradient in the culture medium of nutrients such as cytokines, dissolved oxygen, metabolites and other substances, as well as pH values;
[0007] 2) Various parameters of the static culture system are generally difficult to monitor and control online;
[0008] 3) Operations such as manual fluid change are required, increasing the risk of contamination;
[0009] 4) The two-dimensional static culture is far away from the three-dimensional growth environment in the human body, which is easy to cause the differentiation of stem / progenitor cells to a certain extent
Perfusion (Palsson et al., Bio / Technology, 11, 368, 1993) and fixed-bed bioreactors (Meissner et al., Cytotechnology, 30, 227, 1999) that are often used for co-cultivation of stromal cells and hematopoietic stem cells, due to their internal The fluid shear force is large, the degree of interaction between stromal cells and hematopoietic stem cells is limited, and the effect of expanding hematopoietic stem cells is not good

Method used

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  • Method for expanding hemopoietic stem cell under three-dimensional condition
  • Method for expanding hemopoietic stem cell under three-dimensional condition
  • Method for expanding hemopoietic stem cell under three-dimensional condition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This example is the in vitro culture and identification of mouse neural stem cells embedded in calcium alginate microgel beads.

[0036] In order to verify that cells can grow well in calcium alginate microgel beads, mouse neural stem cells were embedded in calcium alginate microgel beads with a diameter of 2 mm, and cultured and identified in vitro. Specifically, the sixth-generation mouse neural stem cell suspension was prepared, and the total number of cells was 9×10 5 , the cell density was adjusted to 4×10 5 cells mL -1 . Mix this cell suspension with 1.5% sodium alginate solution at a volume ratio of 1:4 (the cell density at this time is 0.8×10 5 cells mL -1 ), through 5 # Needle dropwise to 1.5% CaCl 2 In the solution, react for 10 minutes under stirring conditions, wash with PBS three times, and filter with a sieve to obtain microgel beads. These microgel beads were equally divided into three parts and inoculated into culture flasks containing 3 ml of cul...

Embodiment 2

[0042] This example is the capsule-breaking harvest rate when calcium alginate microgel beads embed mouse calvarial osteoblast cells.

[0043] Calcium alginate microgel beads with a diameter of 2 mm were used to embed mouse calvarial osteoblasts, and the cell harvest rate was determined by dissolving the beads with sodium citrate solution. Specifically, 1 mL of density 6 x 10 5 cells mL -1 Mouse calvarial osteoblasts were mixed with 4 ml of 1.5% sodium alginate solution, passed through 5 # Needle dropwise to 1.5% CaCl 2 solution, reacted for 10 minutes under stirring conditions, and washed three times with PBS. Add 55 mM sodium citrate solution for 10 minutes, centrifuge at 1500 rpm for 10 minutes, discard the supernatant, add 1 ml of medium, pipette, count, and calculate the cell harvest rate.

[0044] The above experiments were repeated three times.

[0045] Experimental results: The cell harvest rates obtained in the three experiments were 86.7%, 79.2%, and 96.4%, resp...

Embodiment 3

[0047] This example is the cultivation of human umbilical cord blood hematopoietic stem cells in a rotating wall bioreactor.

[0048] In order to verify that the RWV culture system can cultivate stem cells that are more sensitive to the growth environment, in this example, human umbilical cord blood hematopoietic stem cells were cultured in the RWV culture system.

[0049] Isolation of mononuclear cells from human umbilical cord blood: umbilical cord blood comes from healthy mothers who gave birth at term or infants born at term by caesarean section, and the average amount of raw blood collected each time is 50-100mL. After cord blood collection, use a density of 1.077g·mL -1 Ficoll lymphocyte separation medium density gradient centrifugation (2500r / min, 25min), centrifugation to obtain mononuclear cells (containing about 1% CD34 + Cells) were then centrifuged and washed twice with IMDM basal medium (1000rpmin, 5min) for later use.

[0050] Preparation of the bioreactor befo...

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Abstract

The invention discloses an enlargement method of hemapoietic stem cell in the three-dimensional condition, which is characterized by the following: using calcium alginate micro-gel ball to embed the trophocytes; proceeding the together culture of embedded trophocytes and hemapoietic stem cell in the rotary wall-pattern reactor; obtaining the hemapoietic stem cell and trophocytes. The invention seperates two-source cell, which reduces the cutting force for hemapoietic stem cell effectively.

Description

technical field [0001] The invention belongs to the fields of biotechnology and tissue engineering, in particular to a method for expanding hematopoietic stem cells under three-dimensional conditions. Background technique [0002] Cells are in a complex growth environment in the body, and there are important connections between different types of cells. Some cells need to rely on other cells to nourish and support them to varying degrees when cultured in vitro. The former are called trophoblast cells. The latter are called trophoblasts. This trophic effect is particularly important for the in vitro culture of stem cells, such as the in vitro culture of hematopoietic stem cells. [0003] Hematopoietic stem cells have broad clinical application prospects. They can rebuild the hematopoietic system of patients after high-dose radiotherapy and chemotherapy, perform gene therapy and immunotherapy, and prepare mature blood products. However, in practical applications, there is a ...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/0789
Inventor 刘天庆李香琴刘洋崔占峰孙相彧马学虎
Owner DALIAN UNIV OF TECH
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