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Serum-free full-suspension domestication method for Sf9 cells

A full-suspension, serum-free technology, applied in biochemical equipment and methods, animal cells, invertebrate cells, etc., can solve the problems of cell activity influence, ordinary medium can not achieve the optimal effect, etc., and achieve high cell viability , high success rate and low initial concentration

Active Publication Date: 2020-09-29
ZHAOQING INST OF BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After research, it is found that for different cells, different media are required, and in many cases, a single common media suitable for the cells may not achieve the optimal effect
[0006] The method of gradually reducing serum can be applied to the acclimation process of many suspension serum-free cell cultures, but the choice of medium will have a significant impact on the acclimatization process and the activity of cells after acclimatization

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Step 1: Adhesive culture of Sf9 cells until the confluency reaches over 90%, discard the Grace's Insect Medium (adherence medium), wash 2 times with 5mL PBS, drain the PBS, and suck the square bottle with an electric pipette Remove the residual liquid at the bottom to completely remove the serum that affects the trypsin digestion.

[0035] Step 2: Add 2 mL of trypsin, shake well and let it stand for 1 min. After the digestion is complete, add 1 mL of FBS to stop the digestion;

[0036] Step 3: blow and blow with Sf-900 III SFM medium, and centrifuge the blown and blown Sf9 cells at 1200 rpm for 4 minutes.

[0037] Step 4: Discard the supernatant of the centrifuged product in step 3, resuspend in 5 mL of Sf-900 III SFM medium, take 200 μL of the suspension and count it with a Counterstar counter, and use Sf-900 III SFM medium for cell density according to the counting results Dilute to 0.6-1.0×10 6 cells / mL, with a volume of 27 mL, transferred to a 125 mL Corning shake...

Embodiment 2

[0046] Generally with reference to Example 1, its concrete steps are as follows:

[0047] Step 1: When the Sf9 cells adhere to the wall and the confluence rate reaches over 90%, discard the culture medium and wash it twice with 5mL PBS. Enzymatically digested serum.

[0048] Step 2: Add 2 mL of trypsin, shake well and let it stand for 2 minutes. After the digestion is complete, add 1 mL of FBS to stop the digestion;

[0049] Step 3: blow and blow with Sf-900 III SFM medium, and centrifuge the blown and blown Sf9 cells at 1200 rpm for 4 minutes.

[0050] Step 4: Discard the supernatant of the centrifuged product in step 3, resuspend in 5 mL of Sf-900 III SFM medium, take 200 μL of the suspension and count it with a Counterstar counter, and dilute the cell density to 0.6-1.0×10 according to the counting result 6 cells / mL, with a volume of 27 mL, transferred to a 125 mL Corning shaker flask, added 3 mL of heat-inactivated fetal bovine serum, and cultured in a shaker at 28°C and...

Embodiment 3

[0058] Generally with reference to Example 1, its concrete steps are as follows:

[0059] Step 1: When the Sf9 cells adhere to the wall and the confluence rate reaches over 90%, discard the culture medium and wash it twice with 5mL PBS. Enzymatically digested serum.

[0060] Step 2: Add 2 mL of trypsin, shake well and let it stand for 2 minutes. After the digestion is complete, add 1 mL of FBS to stop the digestion;

[0061] Step 3: blow and blow with Sf-900 III SFM medium, and centrifuge the blown and blown Sf9 cells at 1200 rpm for 4 minutes.

[0062] Step 4: Discard the supernatant of the centrifuged product in step 3, resuspend in 5 mL of Sf-900 III SFM medium, take 200 μL of the suspension and count it with a Counterstar counter, and dilute the cell density to 0.6-1.0×10 according to the counting result 6 cells / mL, with a volume of 27 mL, transferred to a 125 mL Corning shaker flask, added 3 mL of heat-inactivated fetal bovine serum, and cultured in a shaker at 28°C and...

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Abstract

The invention belongs to the field of biology, and discloses a serum-free full-suspension domestication method for Sf9 cells. The method comprises the following steps: step 1, resuspending the Sf9 cells in an Sf-900 III SFM culture medium added with dextran sulfate, and diluting the Sf9 cells to 0.6-1.0x10<6> cells / mL; step 2, adding a heat-inactivated fetal calf serum to the product in the step 1, and culturing the Sf9 cells; and step 3, repeating the step 1 and the step 2 when the density of the Sf9 cells in the step 2 reaches 4x10<6> cells / mL or above in the third day, wherein the amount ofthe added heat-inactivated fetal calf serum is gradually reduced every time the step 1 and the step 2 are repeated until the density of the Sf9 cells still reaches 4x10<6> cells / mL or above without adding the heat-inactivated fetal calf serum, and the final concentration of dextran sulfate in the Sf-900 III SFM culture medium in the step 1 is 20-30 mg / L. The method has the advantages of low domestication frequency, high concentration of the domesticated cells after proliferation, and high survival rate.

Description

technical field [0001] The invention relates to the field of biology, in particular to a serum-free full-suspension domestication method of Sf9 cells. Background technique [0002] The current mature cell culture methods are mainly monolayer static adherent culture and adsorption culture. Monolayer static adherent culture refers to the culture in which cells are attached to a certain solid surface. The advantages are easy and cheap culture, the infection process is easy to observe under an inverted microscope and all cells can be maintained in culture by this method. The disadvantage is that the viability is low during subculture, and the monolayer cells limit the cell density per milliliter, which affects protein production. Adsorption culture is a kind of immobilized culture method, which is a culture method in which cells are attached to dextran microcarriers, and cells use the surface of microcarriers for adherent growth. Compared with the static culture method, micro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C12N5/02
CPCC12N5/0601C12N2500/84C12N2500/90C12N2501/90C12N2527/00
Inventor 郑航辉方倪冉叶俊贤董楠杨小云
Owner ZHAOQING INST OF BIOTECHNOLOGY CO LTD
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