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Separation method of human intervertebral disc PROCR < + > nucleus pulposus progenitor cells

A separation method and intervertebral disc technology, applied in the field of isolation of human intervertebral disc PROCR+ nucleus pulposus progenitor cells, can solve the problems of simple cell components in the nucleus pulposus, complex induction technology, and remote clinical application

Pending Publication Date: 2022-03-01
中国人民解放军陆军特色医学中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies believed that the cell components in the nucleus pulposus were simple, and only notochord cells and chondrocytes existed. These cells faced practical problems such as difficult acquisition and expansion or complicated induction techniques, and they were still far away from clinical application.

Method used

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  • Separation method of human intervertebral disc PROCR &lt; + &gt; nucleus pulposus progenitor cells
  • Separation method of human intervertebral disc PROCR &lt; + &gt; nucleus pulposus progenitor cells
  • Separation method of human intervertebral disc PROCR &lt; + &gt; nucleus pulposus progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1 solution preparation

[0083] 1. 1× red blood cell lysate:

[0084] with ddH 2 Dilute 10× red blood cell lysate with O and store at 4°C

[0085] 2. Configure 0.2% (w / v) of Pronase:

[0086] 0.1 g was added to 50 mL of distilled water, and the solution was filtered with a 0.22-μm membrane filter, and stored at 4°C.

[0087] 3. Prepare 0.1% (w / v) collagenase II:

[0088] Weigh 0.05g of collagenase II, add 50mL of serum-free DMEM / F12, filter the solution with a 0.22-μm membrane filter, and prepare it for immediate use.

[0089] 4. Prepare 2% penicillin and streptomycin in PBS:

[0090] Take 1mL penicillin and streptomycin and 49mL PBS with a pipette gun, mix well, and store at 4°C.

[0091] 5. Prepare PBS containing 3% BSA:

[0092] Pipette 1.5mL BSA into 48.5mL PBS, mix well, and store at 4°C.

[0093] Embodiment 2 intervertebral disc cell digestion

[0094] 1. Sample acquisition:

[0095] Avoid electrocoagulation of disc tissue during disc surgery ...

Embodiment 3

[0110] Example 3 Cell Quality Inspection

[0111] 1. AO / PI staining:

[0112] Add 1 mL of 2% penicillin and streptomycin solution in PBS to resuspend the cells, pipette 10 ul of the cell suspension and 10 ul of the AO / PI solution to mix at a ratio of 1:1, and stain for 2 minutes.

[0113] 2. Cell count:

[0114] Aspirate 20ul of the stained cell suspension and count with a Counter Star counter.

[0115] The result is as figure 1 As shown, the measured cell viability was 81.35%, and the number of cells was 1.01×10 6 , the light microscope photos show that the background is not clear, the color of the cell pellet is not uniform, and there are many impurities. Magnetic bead sorting is required to remove impurities.

Embodiment 4

[0116] Example 4 Magnetic bead sorting to remove impurities

[0117] 1. Preparation of reagents

[0118]Prepare 1×PBS containing 0.04% BSA, store at 4°C

[0119] ddH 2 O Prepare 1×Binding Buffer

[0120] 2. Remove tissue impurities

[0121] 1) Separation column selection:

[0122] Miltenyi Biotec MS columns require the total number of cells to be 2×10 8 Within, and the number of dead cells is 1×10 7 Within; Miltenyi Biotec LS columns require the total number of cells to be within 2×10 9 Within, and the dead cells are within 1×10 8 within.

[0123] 2) Take no more than 1×10 7 dead cells and 2×10 8 Within the total number of cells, centrifuge at 4°C at 300g / 5min to collect the cells and discard the supernatant.

[0124] 3) Resuspend the cells with 100ul Dead Cell Removal Micro Beads, mix gently and incubate at room temperature for 15min.

[0125] 4) Adsorb the MACS Separator to the MACS MultiStand bracket, select a suitable type of column to adsorb on the separator, ...

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Abstract

The invention provides a method for separating human intervertebral disc PROCR < + > nucleus pulposus progenitor cells, which comprises the following steps of: 1) fully cutting nucleus pulposus into pieces, and sequentially and respectively digesting the nucleus pulposus pieces with trypsin, streptavidin and type II collagenase to obtain a cell suspension; 2) carrying out erythrocyte splitting decomposition treatment on the cell suspension, carrying out magnetic bead sorting to remove tissue impurities, and resuspending cells with PBS + 0.04% BSA to obtain a cell resuspension; and 3) dyeing the cells in the cell resuspension with PROCR flow cytometry antibodies, sorting through a flow cytometry, comparing the sorting efficiency of two mainstream PROCR clone number antibodies, optimizing the sorting scheme, and finally obtaining the human intervertebral disc PROCR + nucleus pulposus progenitor cells. The invention also provides the human intervertebral disc PROCR < + > nucleus pulposus progenitor cell obtained according to the separation method and application thereof. The nucleus pulposus PROCR < + > cells prepared by the invention have good clone formation, self-renewal and three-line differentiation capacities, and a brand new seed cell source is provided for a biological treatment strategy of intervertebral disc degenerative diseases.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to stem cell culture technology, in particular to a human intervertebral disc PROCR + Methods for isolation of nucleus pulposus progenitor cells. Background technique [0002] Degenerative disc disease (Degenerative Disc Disease, DDD) is a high-incidence disease with degeneration of the nucleus pulposus as the core pathological event. The resulting low back pain, neck and shoulder pain, and even paralysis seriously affect the patient's working ability and quality of life. With the aging of the social population, the disability rate and related costs of neck and back pain are increasing year by year. According to the latest WHO statistics, the disability-adjusted life year (DALY) ranking of global diseases, low back pain and neck pain rank first and third place. Therefore, research on the pathogenesis and treatment strategies of intervertebral disc degenerative diseases has im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0669A61K35/28A61P19/08G01N15/14C12N2509/00C12N2509/10
Inventor 甘翼搏刘鹏胡欧朱军晏蒲林黄莎林鹏
Owner 中国人民解放军陆军特色医学中心
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