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Preparation method and application of immortalized human CD34 <+>CD38<-> hematopoietic stem cell line

A CD38-, hematopoietic stem cell technology, applied in the field of preparation of immortalized human CD34+CD38- hematopoietic stem cell lines, can solve the problems of unsuitable candidate cells, complex genetic background, high carcinogenicity, etc., and achieve strong long-term hematopoietic reconstruction ability , The effect of single genetic background and high controllability

Pending Publication Date: 2021-08-31
苏天生命科技(苏州)有限公司
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  • Abstract
  • Description
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Problems solved by technology

But they did not acquire and grow immortal CD34 + CD38 - hematopoietic stem cells
Moreover, the cells they obtained through iPSC have the potential to be highly carcinogenic due to the introduction of the oncogene c-Myc
[0004] In 2017, Changchun Litai Biotechnology Co., Ltd. applied for a patent "Hypoxia Induced Directed Differentiation of Hematopoietic Stem Cells into Immortal Erythroid Progenitor Cells". Stress response of blood cells, making them unsuitable as candidates for hematopoietic stem cell transplantation
More importantly, the cells obtained by the above techniques are multi-group mixed cells, and this group of cells with low purity has a complex genetic background
There will be more unknown potential dangers for itself or the downstream cells derived from differentiation

Method used

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  • Preparation method and application of immortalized human CD34 &lt;+&gt;CD38&lt;-&gt; hematopoietic stem cell line
  • Preparation method and application of immortalized human CD34 &lt;+&gt;CD38&lt;-&gt; hematopoietic stem cell line
  • Preparation method and application of immortalized human CD34 &lt;+&gt;CD38&lt;-&gt; hematopoietic stem cell line

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preparation example Construction

[0055] An immortalized human CD34 + CD38 - A method for preparing a hematopoietic stem cell line, comprising the following steps:

[0056] S1. Isolation of primary CD34 + CD38 - Hematopoietic stem cells, the specific steps are:

[0057] S1.1. Slowly add human whole bone marrow cell suspension to the upper layer of ficoll solution of equal volume, and perform density gradient centrifugation;

[0058] S1.2. Absorb the intermediate white cell layer after gradient density centrifugation, add it to the medium containing 10% FBS or PBS solution, and centrifuge to obtain the cell pellet;

[0059] S1.3. Repeat the operation of S1.2 2 to 3 times;

[0060] S1.4. Incubate the obtained cells with CD34 and CD38 fluorescent antibodies, and obtain CD34 by flow cytometry + CD38 - cell;

[0061] S1.5. CD34 obtained by sorting + CD38 - Cells were cultured in stem cell medium containing SCF / FLT3L / IL6 / G-CSF / IL3 / TPO / UM171 / SR1;

[0062] S2. Obtain CD34 with high expression of HPV16 E6 / E7...

Embodiment

[0075] Describe in detail with a specific embodiment of the present invention below, its specific steps are as follows:

[0076] S1. Isolation of primary CD34 + CD38 - Hematopoietic stem cells, the specific steps are:

[0077] S1.1. Slowly add the human whole bone marrow cell suspension to the upper layer of an equal volume of ficoll solution, and perform density gradient centrifugation. The centrifugation condition is 800g, 30 minutes, and slowly accelerates and decelerates;

[0078] S1.2. Absorb the intermediate white cell layer after gradient density centrifugation, add it to the medium containing 10% FBS or PBS solution, and centrifuge to obtain the cell pellet. The centrifugation condition is 2000rpm for 5 minutes;

[0079] S1.3. Repeat the operation of S1.2 2 to 3 times;

[0080] S1.4. Incubate the obtained cells with CD34 and CD38 fluorescent antibodies, see figure 1 As shown, CD34 was obtained by flow sorting + CD38 - cell;

[0081] S1.5. CD34 obtained by sortin...

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Abstract

The invention discloses a preparation method and application of an immortalized human CD34 <+>CD38<-> hematopoietic stem cell line. The method comprises the following steps of: separating a primary CD34 <+>CD38<-> hematopoietic stem cell, then, obtaining the CD34 <+>CD38<-> hematopoietic stem cell which expresses HPV16E6 / E7, finally, obtaining a CD34 <+>CD38<-> monoclonal cell which expresses the HPV16E6 / E7, and carrying out passage and cryopreservation on the successfully-proliferated immortalized CD34 <+>CD38<-> monoclonal cell. Compared with a CD34 hematopoietic stem cell line established in the past, the CD34 <+>CD38<-> hematopoietic stem cell line prepared by the invention has better hematopoietic stem cell characteristics, has better long-range hematopoietic reconstitution potential, is the monoclonal cell, has a single genetic background, has higher controllability, and hopefully provides continuous donor cells for hematopoietic stem cell transplantation, and a possibility of in vitro acquisition of artificial component blood and artificial whole blood is improved.

Description

technical field [0001] The invention belongs to the technical field of hematopoietic stem cell line construction, in particular to an immortalized human CD34 + CD38 - Preparation method and application of hematopoietic stem cell line. Background technique [0002] With the increasing demand for safe blood worldwide, international and domestic counterparts are trying to produce artificial blood in vitro. Hematopoietic stem cells are the source of all functional blood cells, but the number of hematopoietic stem cells is scarce, accounting for less than one ten-thousandth of the total bone marrow, making it difficult to effectively proliferate and passage in vitro. Express CD34 only + The cell line of the same type has a heterogeneous cell population composition, mainly hematopoietic progenitor cells rather than hematopoietic stem cells. CD34 + Stem cell lines to establish CD34 that can be cultured in vitro + CD38 - Hematopoietic stem cell lines are more meaningful, but ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789C12N15/867C12N15/37C12N5/10C12R1/91
CPCC12N5/0647C12N15/86C07K14/005C12N2740/15043C12N2800/107C12N2710/20022C12N2510/04
Inventor 王建荣方艺璇李磊
Owner 苏天生命科技(苏州)有限公司
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