Storage liquid for mammalian cells

A mammalian and preservation solution technology, applied in the field of mammalian cell preservation solution, can solve the problem of PD-1 expression reduction and other issues

Pending Publication Date: 2021-10-26
MEGAKARYON CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it was reported that after T cells were cryopreserved, the expression of PD-1 (immune checkpoint molecule) on the T cell membrane was significantly reduced (Non-Patent Document 3), and it is necessary to develop a non-cryopreservation method for T cells

Method used

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  • Storage liquid for mammalian cells
  • Storage liquid for mammalian cells
  • Storage liquid for mammalian cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] [Production of platelets derived from iPS cells]

[0079] Platelets derived from iPS cells were prepared according to the method described in PCT / JP2018 / 034667. The specific procedure is shown in (1-1) to (1-12) below.

[0080] (1-1) Preparation of hematopoietic precursor cells from iPS cells

[0081]According to the method of Takayama et al. (J.Exp.Med., 2010, vol.13, 2817-2830), from human iPS cells (TKDN SeV2 and NIH5: iPS derived from human fetal skin fibroblasts established using Sendai virus) cells) to differentiate into blood cells. Specifically, human ES / iPS cell colonies were co-cultured with C3H10T1 / 2 feeder cells in the presence of 20 ng / mL VEGF (manufactured by R&D SYSTEMS) for 14 days to produce hematopoietic progenitor cells (HPCs). . The above culture was carried out at 37°C, 20% O 2 , 5%CO 2 implemented under the conditions.

[0082] (1-2) Gene introduction system

[0083] The gene transfer system utilizes a lentiviral vector system. The lentivi...

Embodiment 2

[0142] [Add vitamin C to the platelet storage solution]

[0143] (2-1) Preparation of platelet preservation solution

[0144] Bicarbonate Ringer's solution (BICANATE infusion solution; manufactured by Otsuka Pharmaceutical Factory, Inc.) (sodium chloride 5.84 g / L, potassium chloride 0.30 g / L, calcium chloride hydrate 0.22 g / L, magnesium chloride 0.20g / L, sodium bicarbonate 2.35g / L, and sodium citrate hydrate 0.20g / L), add human serum albumin preparation (HSA; CSL Behring company manufacture), and blood preservation solution (ACD-A liquid; Terumo Co., Ltd.) (2.20 W / V% of sodium citrate hydrate, 0.80 W / V% of citric acid hydrate, and 2.20 W / V% of glucose). In this specification, this solution is also referred to as "first-generation preservation solution". Separately, a solution obtained by adding VC preparation (injection preparation containing additives; manufactured by Sawai Pharmaceutical Co., Ltd.) to the first-generation preservation solution was prepared. In this specif...

Embodiment 3

[0160] [Add VC and VB3 to platelet preservation solution]

[0161] (3-1) Preparation of platelet preservation solution

[0162] A solution was prepared by adding nicotinic acid (niacin injection preparation; manufactured by TOA EIYO Co., Ltd.) to the VC-added preservation solution. It should be noted that, as mentioned above, in the specification of this application, "VB3" means nicotinic acid and / or nicotinamide. Niacin is used as VB3 in Examples 3-5 and 8-13, and nicotinic acid is used as VB3 in Example 6. acid or nicotinamide as VB3. In addition, in this specification, a solution obtained by adding VB3 (nicotinic acid or nicotinamide) to a VC-added storage solution is also referred to as a "VC / VB3-added storage solution" or a "second-generation storage solution". The final concentrations of the above-mentioned additives in each preservation solution are shown in Table 3 below. In addition, any preservation solution was adjusted to pH 7.3±0.1 using 1M NaOH, and incubated ...

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Abstract

The present invention addresses the problem of providing a novel storage liquid for storing mammalian cells over a long period of time in a non-frozen state. Ascorbic acid and / or niacin are added to an isotonic liquid to give a storage liquid. Then the storage liquid is mixed with platelets and stored under shaking. Thus, deterioration in the functions of the platelets and a decrease in the survival rate thereof can be prevented for at least 10 days. When the aforesaid storage liquid is mixed with mesenchymal stem cells, megakaryocytes or T cells and stored in a non-frozen state, deterioration in the functions of these cells and a decrease in the survival rate thereof can be prevented for at least from several days to several ten days.

Description

technical field [0001] The present invention relates to a preservation solution of mammalian cells containing niacin or its derivatives or their salts (hereinafter sometimes collectively referred to as "niacins") and antioxidants, and a powder for preparing the preservation solution . A preservation method for mammalian cells using the preservation solution. Background technique [0002] Platelet preparations are administered to patients with thrombocytopenia in addition to administration of bleeding due to surgery or trauma. At present, platelet preparations are manufactured from blood obtained through blood donation, but there are concerns that the number of blood donations will decrease due to changes in population structure, and there will be a shortage of platelet preparations. [0003] In addition, when a donor of blood donation suffers from an infectious disease such as a bacterium, the blood may be contaminated by the bacterium, so there is a risk of causing an infe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/078C12N5/0783C12N1/04
CPCC12N1/04C12N5/0644C12N2506/45C12N2501/606C12N2501/60C12N2500/38C12N5/0663C12N5/0636C12N5/0662C12N5/0622
Inventor 富冢順子重盛智大渡边奈月竹绳太一白川智景西村益浩藤田泰毅泽本修
Owner MEGAKARYON CORP
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