Preparation method of lung hardness base in vitro cell culture platform

A technology of in vitro cell culture and basal medium, applied in the field of bioengineering, can solve problems such as unsatisfactory

Active Publication Date: 2019-12-31
惠森生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a method for preparing a lung stiffness base in vitro cell culture platform. The preparation method of the lung stiffness base in vitro cell culture platform should solve the problem of in vitro cell culture in the prior art. Unsatisfactory technical problems of cell culture platform in simulating <1KPa low hardness organ tissue

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  • Preparation method of lung hardness base in vitro cell culture platform
  • Preparation method of lung hardness base in vitro cell culture platform
  • Preparation method of lung hardness base in vitro cell culture platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of in vitro cell culture platform with different lung stiffness substrates

[0034] Specific steps are as follows:

[0035] 1. Using lentivirus-mediated overexpression technology to form lentivirus packaging LOXL2 overexpression plasmid (LV-LOXL2-OE), infect liver cancer cell MHCC97H, first with 3ug / mL puromycin, volume percentage concentration is 10% FBS and volume The DMEM culture solution with a percentage concentration of 1% penicillin and streptomycin was used for positive selection of infected cells until the cells did not die obviously, and then the medium was changed with DMEM containing 10% FBS and 1% penicillin and streptomycin In culture, the cells grow to about 90% density. Cells were collected to determine the degree of LOXL2 overexpression. Subsequently, liver cancer cells overexpressing LOXL2 at a density of 90% were cultured in DMEM serum-free medium for 24 hours, and the supernatant was collected (CM-LV-LOXL2-OE);

[0036] Spec...

Embodiment 2

[0044] Example 2 Cultivating Human Lung Fibroblast HELF Cells Using Different Lung Basal Stiffness Cell Culture Platforms

[0045] Human lung fibroblast HELF was purchased from the Institute of Biochemical Cells, Chinese Academy of Sciences. It was cultured with DMEM medium containing 10% FBS by volume and 0.5% penicillin and streptomycin. The cells grew to a density of about 90%. The collected cells were digested with 0.25% trypsin.

[0046] The specific steps for culturing human lung fibroblasts on the substrate platforms with different lung stiffness prepared above are as follows:

[0047] 1. Make the collected cells into a cell suspension about 3×10 6 Cells / ml culture medium;

[0048] 2. Take 2ml of cell suspension respectively, and gently drop on the cell culture platform;

[0049] 3. Cultivate in a cell incubator at 37°C and 5% carbon dioxide for 24 hours;

[0050] 4. Observe the changes in cell morphology.

[0051] Experimental results (such as image 3 ) showed t...

Embodiment 3

[0052] Example 3 Effects of High and Low Lung Stiffness Substrates on the Expression of Genes Associated with Matrix Remodeling in Lung Fibroblasts

[0053] The human lung fibroblasts cultured in Example 2 were collected, and the expression of matrix remodeling-associated genes (MMP2, MMP9, Fibronectin), chemokine CXCL12 and activation of related pathways in the lung fibroblasts were detected. It was found that (such as Figure 4 A, 4B, 4C, 4E), LOXL2 can significantly up-regulate the expression of matrix remodeling-associated genes in lung fibroblasts, and the combined effect of LOXL2 and substrate stiffness can further enhance the expression of matrix remodeling-associated genes in lung fibroblasts, suggesting that LOXL2 has important Ability to model lung metastatic target organ matrix.

[0054] At the same time, both CM-LV-LOXL2-OE supernatant and pure LOXL2 can activate the AKT pathway and promote the expression of Fibronectin, and inhibit the phosphorylation level of AK...

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Abstract

The invention provides a preparation method of a lung hardness base in vitro cell culture platform. The preparation method comprises the steps of mixing rat tail I type collagen with DMEM basic culture mediums in different volume ratios, and performing placing in a first perforated plate; mixing the rat tail I type collagen with DMEM basic culture fluid and CM-LV-LOXL2-OE in the volume ratio being1 to 10 to 5, and performing placing in a second perforated plate; putting the first perforated plate and the second perforated plate in an incubator of 37 DEG C for standing; and placing a base after colloid formation in a refrigerator of 4 DEG C, and after 10-14h, forming an in vitro cell culture platform having different base hardness. Through the adoption of the preparation method disclosed by the invention, the hardness changes of in vivo low-hardness internal organ tissue of which the hardness is smaller than 1KPa can be well simulated, and a novel in vitro research platform is providedfor seeking the general molecular characteristics of entity tumorigenesis pulmonary metastasis and discussing a pathomechanism about transferring target organs to be re-modeled and reformed into appropriate soil.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a cell culture technology, in particular to a preparation method of an in vitro cell culture platform simulating the hardness of lung tissue in vivo and its application in the study of the formation mechanism of pre-transfer niches in the process of tumor lung metastasis. Background technique [0002] A large number of clinical data show that many solid tumors, including liver cancer, esophageal cancer, and breast cancer, are prone to lung metastasis. Finding the common molecular characteristics of lung metastasis of solid tumors and exploring the relevant pathological mechanisms of the remodeling of metastatic target organs into "suitable soil" before tumor colonization and metastasis have gradually become new growth points in current tumor metastasis research. An ideal in vitro research platform that reflects the characteristics of lung tissue stiffness will undoubtedly promote the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0688C12N2533/54C12N2533/90
Inventor 邢晓侠吴思凡崔杰峰张希
Owner 惠森生物科技(上海)有限公司
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