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Serum-free cryoprotectits agent

A low-temperature protective agent and serum-free technology, applied in the field of cell culture, can solve the problems of difficult serum removal, complex serum components, and unclear components of serum-free low-temperature storage reagents, etc., and achieve the effect of clear components and easy preparation

Inactive Publication Date: 2013-07-24
四川全组生命科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] One of the objectives of the present invention is to provide a serum-free cryoprotectant, which aims to solve the problems of complex serum components, difficult removal of serum residues and unclear components of serum-free low-temperature storage reagents in the prior art.

Method used

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Examples

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Effect test

Embodiment 1

[0025] A serum-free cryoprotectant, comprising an intracellular osmotic protectant and an extracellular protectant, in every 100mL of serum-free cryoprotectant, the composition weight of the extracellular protectant: 800mg of NaCl, 20mg of KCL, 20mg of NaCl 2 HPO 4 .12H 2 O is 290mg, KH 2 PO 4 24mg, 5g of PEG (400), 3.423g of D-trehalose, 1000ug of collagen IV, and 5mL of DMSO as the intracellular osmoprotectant.

[0026] A method for preparing serum-free cryoprotectant,

[0027] Step 1: NaCl 800mg, KCL 20mg, NaCl 2 HPO 4 .12H 2 O 290mg, KH 2 PO 4 24 mg, PEG (400) 5 g, and D-trehalose 3.423 g were dissolved in 60 mL of water for injection, stirred until completely dissolved, and the solution was kept at 40°C;

[0028] Step 2: Cool down the solution described in step 1 to 20°C, add 1000ug of collagen IV, stir until it is completely dissolved, set the volume to 95mL, and keep the solution at 20°C;

[0029] Step 3: In a sterile environment, filter and sterilize the 20°C...

Embodiment 2

[0032] A serum-free cryoprotectant, comprising an intracellular osmotic protectant and an extracellular protectant, in every 100mL of serum-free cryoprotectant, the composition weight of the extracellular protectant: 800mg of NaCl, 20mg of KCL, 20mg of NaCl 2 HPO 4 .12H 2 O is 290mg, KH 2 PO 4 24mg, 15g of PEG (2000), 6.846g of D-trehalose, 100ug of collagen IV, and 15mL of DMSO as the intracellular osmoprotectant.

[0033] A method for preparing serum-free cryoprotectant,

[0034] Step 1: NaCl 800mg, KCL 20mg, NaCl 2 HPO 4 .12H 2 O 290mg, KH 2 PO 4 24mg, PEG (2000) 15g, and D-trehalose 6.846g were dissolved in 60mL water for injection, stirred until completely dissolved, and the solution was kept at 40°C;

[0035] Step 2: Cool down the solution described in step 1 to 20°C, add 1000ug of collagen IV, stir until completely dissolved, set the volume to 85mL, and keep the solution at 20°C;

[0036] Step 3: In a sterile environment, filter and sterilize the 20°C heat pre...

Embodiment 3

[0048] A serum-free cryoprotectant, comprising an intracellular osmotic protectant and an extracellular protectant, in every 100mL of serum-free cryoprotectant, the composition weight of the extracellular protectant: 800mg of NaCl, 20mg of KCL, 20mg of NaCl 2 HPO 4 .12H 2 O is 290mg, KH 2 PO 4 24mg, 12g of PEG (2000), 5.256g of D-trehalose, 700ug of collagen IV, and 15mL of DMSO as the intracellular osmoprotectant.

[0049] A method for preparing serum-free cryoprotectant,

[0050] Step 1: NaCl 800mg, KCL 20mg, NaCl 2 HPO 4 .12H 2 O 290mg, KH 2 PO 4 24mg, PEG (2000) 12g, and D-trehalose 5.256g were dissolved in 60mL water for injection, stirred until completely dissolved, and the solution was kept at 40°C;

[0051] Step 2: Cool down the solution described in step 1 to 20°C, add 700ug of collagen IV, stir until it is completely dissolved, set the volume to 85mL, and keep the solution at 20°C;

[0052] Step 3: In a sterile environment, filter and sterilize the 20°C ins...

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Abstract

The invention disclose a serum-free cryoprotectits agent which comprises an intracellular permeating protecting agent and an extracellular protecting agent, wherein, the extracellular protecting agent comprise NaCL, KCL, Na2HPO4.12H2O, KH2PO4, PEG(400 to 2000), D-trehalose and type-IV collagen; and the intracellular permeating protecting agent is DMSO (dimethylsulfoxide). The invention further discloses a method for preparing the serum-free cryoprotectits agent. The serum-free cryoprotectits agent does not contain serum from any source, and serum cannot be left over when cells are stored under low temperature; and in addition, the serum-free cryoprotectits agent has definite and simple components, is easy to prepare, and can be used for storing mesenchymal stem cells under low temperature.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a serum-free cryoprotectant. Background technique [0002] At present, animal cell cryoprotectants prepared in vitro mainly use serum-containing DMSO solution, but the serum residues of cell preparations stored with this cryoprotectant are difficult to remove, and the composition of serum is complex, which limits the use of cell preparations. [0003] Mesenchymal stem cells are different from other adult diploid cells. They are cells with self-renewal and multi-lineage differentiation capabilities. They have a wide range of medical applications. Their low-temperature storage has higher requirements for formulations. In order to meet clinical needs, low-temperature The protective agent preferably does not contain serum components. [0004] Serum-free cryoprotectants are the development direction of mesenchymal stem cell cryoprotectants. The prior art has disclosed the use of PEG or tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
Inventor 苑春慧邓涛戴成祥
Owner 四川全组生命科技有限公司
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