CN104928245A

CN104928245A - Method for quickly amplifying human autologous hematopoietic stem cells in vitro

Info

Publication number: CN104928245A
Application number: CN201510311177.8A
Authority: CN
Inventors: 冯文峰; 黄鹏; 昌芒
Original assignee: Guangzhou Stemove Biotechnology Co Ltd
Current assignee: Guangzhou Stemove Biotechnology Co Ltd
Priority date: 2015-06-09
Filing date: 2015-06-09
Publication date: 2015-09-23

Abstract

The invention provides a method for quickly preparing human autologous hematopoietic stem cells in vitro. The method comprises the following steps: 1, drawing autologous blood; 2, collecting and separating autologous mononuclear cells; 3, cultivating the autologous mononuclear cells in vitro. The cell culture medium adopted in the cultivation of the autologous mononuclear cells in vitro is RPMI1640; an artificially synthesized hemocyte maturation promoting factor is added in the culture medium. The amplification method of human autologous hematopoietic stem cells related to the invention has remarkable advantages in the aspects of yield, purity, production speed and safety.

Description

A kind of method of external rapid amplifying people autologous stem cell

technical field:

The present invention relates to biomedical sector, specifically, relate to cell injuring model field, especially a kind of method of external rapid amplifying people autologous stem cell.

background technology:

Hemopoietic stem cell (Hematopoietic stem cells, HSCs) is the adult stem cell in blood system, is a heterogeneous colony, has the ability of long-term self and is divided into the potential of all kinds of mature blood cell.It is the longest and the most deep class adult stem cell of research history, to study of various stem cell, as tumor stem cell, has great importance.

In decades, hemopoietic stem cell is applied to the treatment of disease in the blood system clinically, is proved to be the effective ways for the treatment of malignant hematologic disease.Hemopoietic stem cell is also proved to be has transdifferentiationof ability, has the potential being differentiated to form the various kinds of cell such as osteocyte, neurocyte and myocardial cell.So autologous stem cell is also widely used in the reproducibility repairing and treating of the various diseases in regenerative medicine and repair medical science field.

But, hemopoietic stem cell is applied to clinically deposits a problem, the medullary cell quantity that single collection process obtains often is not enough to for being transplanted, therefore often repeatedly Bone marrow collection is carried out, the method causes very large damage to patient, and easily produce crossed contamination, cell quantity does not reach clinical criteria needs far away.

Therefore, fast a kind of, effectively, the method for the amplifying candidate stem cell of safety is needed badly and is established.

summary of the invention:

The invention solves the hematopoietic stem cell that increases in vitro (hematopoietic stem cells, HSCs) does not reach requirement subject matter for clinical cytology quantity.Provide a kind of external method preparing autologous stem cell fast.

A kind of method of external rapid amplifying people autologous stem cell:

(1), autohemic extraction; (2), the collection of autologous monocyte and separation; (3), the vitro culture of autologous monocyte.

Further, aseptic aspiration autoblood 100-200ml, density gradient centrifugation collecting monocytic cell, cultivates 6 days by autologous monocyte in cell culture fluid, collects the autologous stem cell generated.

Further, collection and the separation method of described step (2) autologous monocyte are: density gradient centrifugation, and concrete steps are as follows:

1). take from body anticoagulated whole blood 100ml, add equal-volume PBS, fully mix;

2). be dispensed into 4 50ml and respectively add in the centrifuge tube of 12.5ml lymphocyte separation medium (Ficoll) in advance;

3) .700G, the centrifugal 20min of room temperature, slow liter is slow to fall (without breaking);

4). extract tunica albuginea cellular layer, join and be equipped with in the 50ml centrifuge tube of 5ml RPMI1640;

5). add 45ml RPMI1640,260G, 4 DEG C of centrifugal 10min, exhaust supernatant;

6). add 50ml RPMI1640,180G, 4 DEG C of centrifugal 10min, exhaust supernatant;

7). add 50ml RPMI1640,110G, 4 DEG C of centrifugal 10min, exhaust supernatant;

8). add 30ml RPMI 1640, for subsequent use.

Use whizzer for Eppendorf centrifuge 5810 in the collection of described autologous monocyte and separation method: horizontal rotor AT-4-62, centrifugal radius 16cm.

The extracorporeal culturing method of described step (3) autologous monocyte is:

Basic culture solution is RPMI1640, in basic culture solution, add additive, described additive is the hemocyte maturation promoting factor of synthetic, and substratum contains erythropoietin (erythropoietin, EPO) final concentration is 2-20ng/ml, preferably 10 ng/ml.

Culture condition in described cell culture fluid is: temperature is the CO2 of 37 DEG C and 5%.

Further, the external concrete cultural method of autologous monocyte is:

The autologous whole blood of 1.100ml is separated through lymphocyte separation medium, obtains about 1 × 10 ⁸individual mononuclearcell;

2. by cell suspension in 30ml RPMI 1640, cell concn is about 1.5-2.0 × 10 ⁶/ ml, adds final concentration 10 ng/ml EPO, culturing bottle is placed in 37 DEG C, cultivate under 5%CO2 incubator condition;

3. cultivate after 24 hours, add IL-3 1000IU/ml, 10 ng/ml EPO;

4., at 3-4 days, add 30ml fresh RPMI 1640(cumulative volume 60ml), and add IL-6 1000IU/ml, 10 ng/ml EPO;

5., at 5-6 days, add 60ml RPMI 1640(cumulative volume 120ml), and add 10 ng/ml EPO;

6. the 7th day bacterial detection fungus culture is negative; Placenta blue staining tests viable cell > 95%, and collecting cell suspension is centrifugal remove supernatant after, more namely obtain the hemopoietic stem cell of high density for 2 times with brine.

IL-3 1000IU/ml in described step 3,10 ng/ml EPO can add in step 2.

beneficial effect:

The present invention is by the regulation and control to the key regulator in hemopoietic stem cell proliferation process, erythropoietin (erythropoietin is added in substratum, EPO), provide a kind of method of external rapid amplifying autologous stem cell, described method is simple, fast, effectively, security is high.The method of the invention is intended adopting and is increased to the HSCs of health in the lab, and donor only need provide the hemopoietic stem cell of less amount in body, and it is for hematopoietic stem cell is frozen and storage provides feasibility basis simultaneously.

accompanying drawing illustrates:

Fig. 1: income earner's autologous stem cell CD34 specific phenotypes qualification figure after human blood mononuclearcell epo protein rapid in-vitro propagation, before (A) propagation, after (B) propagation.

embodiment:

Embodiment 1

(1) all reagent and substratum (RPMI 1640+10 ng/ml EPO+1000IU/ml IL-6) are all aseptically, operate at the Biosafety operator's console of 1,000,000 grades of cleanliness factors, prepare under low temperature (4-10 DEG C) condition, the membrane filtration through 0.22 micron pore size is made.

(2) gather healthy human blood 100ml, use density gradient centrifugation method to be separated and obtain blood mononuclear cells.By the mononuclearcell of acquisition with 5 × 10 ⁶the density of individual/ml, is inoculated in Tissue Culture Flask, and adds the cell culture fluid of preparation in step (1) wherein, 37 DEG C and 5% CO ₂incubator in cultivate 7 days thus obtain the autologous stem cell of epo protein rapid induction.

(3) mensuration of gained autologous stem cell transformation efficiency

1. the CD34+ cell yield of cells were tested by flow cytometry gained autologous stem cell

2. income earner's autologous stem cell is moved on in EP pipe, centrifugal 5min under 1024 revs/min of lower normal temperature.

3. remove supernatant liquor, often pipe adds confining liquid (2%FCS, 1%SA PBS dilutes) the resuspension cell precipitation of 400 μ l.

4. in EP pipe, add the CD34 monoclonal antibody reagent of the fluorized marking of 20 μ l.Be placed on dark place after mixing and hatch 30min, after carry out flow cytometry analysis, automatically calculate the CD34 positive cell quantity in sample and per-cent.The results are shown in Figure 1.

Claims

1. a method for external rapid amplifying people autologous stem cell, comprising:

(1), autohemic extraction; (2), the collection of autologous monocyte and separation; (3), the vitro culture of autologous monocyte, the collection of described autologous monocyte and be separated and adopt density gradient centrifugation, the substratum that the vitro culture of described autologous monocyte uses is RPMI1640.

2. the method for a kind of external rapid amplifying people autologous stem cell as claimed in claim 1, is characterized in that: the hemocyte maturation promoting factor also adding synthetic in described substratum.

3. the method for a kind of external rapid amplifying people autologous stem cell as claimed in claim 2, is characterized in that: the hemocyte maturation promoting factor of described synthetic, containing erythropoietin final concentration 2-20ng/ml.

4. the method for a kind of external rapid amplifying people autologous stem cell as claimed in claim 3, is characterized in that: described erythropoietin final concentration is 10 ng/ml.