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Process for coculturing cord blood hematopoietic stem cells and mesenchymal stem cells in dynamic suspending condition

A hematopoietic stem cell and co-cultivation technology is applied in the field of co-cultivation of cord blood hematopoietic stem cells and mesenchymal stem cells, which can solve the problems of high-dose cytokine combination being expensive, and achieve the effect of being beneficial to expansion and reducing shear force.

Inactive Publication Date: 2008-10-15
DALIAN UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Chen et al. (Chen et al., Stem Cells, 2006, 24(9): 2052-2059) used RWVB to achieve the co-culture of bone marrow-derived HSCs and MSCs under the condition of adding a combination of high-dose cytokines, although the HSCs Expanded 9-fold and 29-fold with MSCs, but high-dose cytokine combinations are obviously more expensive
The in vitro co-culture of HSCs and MSCs derived from cord blood has not been reported so far

Method used

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  • Process for coculturing cord blood hematopoietic stem cells and mesenchymal stem cells in dynamic suspending condition
  • Process for coculturing cord blood hematopoietic stem cells and mesenchymal stem cells in dynamic suspending condition
  • Process for coculturing cord blood hematopoietic stem cells and mesenchymal stem cells in dynamic suspending condition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] In this example, UCB-HSCs and UCB-MSCs were co-cultured under static conditions.

[0027] The freshly isolated UCB-MNCs were divided into 5×10 5 cells mL -1 The density was inoculated into a six-well plate covered with GCSC microcarriers in advance, mixed evenly with calcium alginate chitosan beads embedding ADSCs, and the inoculation density of ADSCs was 1×10 5 cells mL -1 , add IMDM medium to make the culture system 4mL, 37℃, 5%CO 2 , 20%O 2 , Co-cultivated for 9 days under saturated humidity conditions. The concentration of microcarriers was 5 mg·mL -1 , cytokine combination and dosage: SCF 15ng·mL -1 , FL 5ng·mL -1 , TPO 6ng·mL -1 , IL-315ng·mL -1 , G-CSF 1ng·mL -1 , GM-CSF 5ng·mL -1 . NCs were counted every 24 hours, and 2 mL of cell suspension containing GCSC microcarriers were taken from the culture system at 72 hours and 216 hours, digested with 0.125% trypsin containing 0.02% EDTA, and flowed Cytometry analysis and detection of CD34 + CD45 + CD10...

Embodiment 2

[0031] In this example, UCB-HSCs and UCB-MSCs are co-cultured under dynamic suspension conditions.

[0032] The freshly isolated UCB-MNCs were 1 × 10 7 cells mL -1 The density was inoculated into a six-well plate covered with GCSC microcarriers in advance and incubated for 24 hours. After UCB-MSCs fully adhered to the microcarriers, they were mixed with calcium alginate chitosan beads embedding ADSCs and supplemented with IMDM. Medium so that the cell density of UCB-MNCs and ADSCs can be 5×10 in dynamic suspension culture, respectively 5 cells mL -1 and 1×10 5 cells mL -1 , the microcarrier concentration was 5 mg·mL -1 , The combination and dosage of cytokines are the same as in Example 1. Inoculate this mixture into RWVB, keep the speed of the culture chamber of the reactor at 6 rpm, 37°C, 5% CO 2 , 20%O 2 , Co-cultivated for 12 days under saturated humidity conditions. NCs were counted every 24 hours, and 4 mL of cell suspension containing GCSC microcarriers were ta...

Embodiment 3

[0036] This example is the colony-forming CFU-Cs culture of expanded UCB-HSCs.

[0037] CFU-Cs colonies mainly include CFU-Mix’s, CFU-GMs and BFU-Es. The suspension cells in RWVB were cultured for CFU-Cs colonies at 0 hour, 72 hours and 216 hours respectively. The expanded UCB-HSCs were divided into 2×10 5 cells mL -1 Inoculate in methylcellulose medium at a density of 1 mL per 35 mm Petri dish, and 3 dishes in parallel for each sample. 37°C, 5% CO 2 , 20%O 2 1. Cultivate in an incubator with saturated humidity for 10 to 12 days, and count the number of colonies with more than 50 cells under an inverted microscope. The medium is IMDM containing 0.9% methylcellulose, supplemented with 30% FBS, 1% BSA, 50 U·mL -1 Penicillin, 50mg·mL -1 Streptomycin, 1×10 -4 M 2-mercaptoethanol, 2mM L-glutyl ammonium, 50ng mL -1 Recombinant SCF, 10ng·mL -1 Recombinant human IL-3, 10ng·mL -1 Recombinant human GM-CSF and 3U·mL -1 Recombinant human EPO.

[0038] Experimental results: UC...

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Abstract

The invention discloses a method for co-culturing cord blood stem cells and mesenchymal stem cells, belonging to the biotechnology and tissue engineering technical field. The method is characterized by adding no blood serum but only cytokine and trophocyte; wrapping the trophocyte calcium alginate-chitosan gel beads; using microcarrier and rotating wall vessel to implement the co-culture of cord blood stem cells and mesenchymal stem cells; and separating and obtaining the cord blood-derived stem cells respectively. The invention has the advantages that: the microcarrier provides a surface for the cord blood mesenchymal stem cells to adhere to, so the adherent, dynamic, suspension culture of the cord blood mesenchymal stem cells can be implemented. The rotating wall vessel provides an environment for hemopoietic stem cell to suspend and growth in, and also reduces the damages caused by fluid shear stress to the hemopoietic stem cells. The throphocyte can nourish the cord blood stem cells outside the gel beads, replace partially blood serum and contribute to the separation and harvest of different cells.

Description

technical field [0001] The invention belongs to the field of biotechnology and tissue engineering, and in particular relates to a method for co-cultivating umbilical cord blood hematopoietic stem cells and mesenchymal stem cells under dynamic suspension conditions. Background technique [0002] Hematopoietic stem cells (HSCs) can be widely used in clinical applications such as gene therapy, tumor purification and bone marrow transplantation, especially hematopoietic stem cell transplantation has become a routine method for the reconstruction of hematopoietic function in cancer patients after chemotherapy Journal of Biochemistry & Cell Biology, 2004, 36(4): 607-620; Masson et al., Stem Cells, 2004, 22(6): 897-907). However, the limited number of cells greatly limits its clinical application. An effective way to solve this problem is to implement large-scale expansion of HSCs in vitro. Although hematopoietic stem cell transplantation can significantly improve the quality of l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12M3/02C12N5/0775C12N5/0789
Inventor 刘天庆李香琴范秀波孙相彧崔占峰马学虎
Owner DALIAN UNIV OF TECH
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