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Method for constructing mammalian esophageal squamous epithelial immortalized cell line, constructed cell line and organoid thereof

A technology of immortalized cell lines and construction methods, which is applied in the field of construction of esophageal organoids and esophageal squamous epithelial immortalized cell lines, which can solve problems such as unfavorable research, cell cycle, and genome stability defects of apoptosis

Pending Publication Date: 2021-12-14
汉姆德(宁波)智能医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the immortalized cells obtained by expressing the large T antigen of SV40 have defects in cell cycle, apoptosis and genome stability, which is not conducive to subsequent research

Method used

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  • Method for constructing mammalian esophageal squamous epithelial immortalized cell line, constructed cell line and organoid thereof
  • Method for constructing mammalian esophageal squamous epithelial immortalized cell line, constructed cell line and organoid thereof
  • Method for constructing mammalian esophageal squamous epithelial immortalized cell line, constructed cell line and organoid thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Establishment of Esophageal Squamous Epithelial Immortalized Cell Line

[0052] 1. Preparation of primary rat squamous epithelial cells

[0053] Anesthetize the rats with 10% chloral hydrate anesthesia at a dose of 0.3ml / 100g intraperitoneally, perform routine surgical disinfection, and prepare instruments; remove the esophagus after sacrificing the rats, rinse in PBS to remove blood stains, and then remove the esophagus along the long axis of the esophagus. The esophagus was cut open; the esophageal muscular layer and esophageal mucosa were torn apart with forceps, and the esophageal epithelial tissue was preserved; in the ultra-clean workbench, the esophageal epithelial tissue was rinsed with sterile PBS, and then transferred to a medium containing 5× double antibody (final concentration 500U / mL penicillin, 500μg / mL streptomycin) in DMEM medium for 10min, and then washed with sterile PBS; put the esophageal epithelial tissue in a petri dish, add 3ml 2.4U / ml ...

Embodiment 2

[0067] Example 2 Characteristics of Immortalized Esophageal Squamous Cell Line

[0068] 1. Cell morphology and origin

[0069] D3, F3 and A1 single-cell clones were further cultured to establish "normal" immortalized rat esophageal epithelial cell lines, and the cell lines formed by D3, F3 and A1 were observed with an optical microscope. The experimental results are as follows: Figure 4 Shown; karyotype analysis as Figure 5 Shown; Immunofluorescent staining results of the suprabasal layer marker CK13 and the basal layer marker CK14 are shown in Figure 6 and Figure 7 as shown, Figure 6 The results of immunofluorescence staining of CK13 and CK14 in normal esophageal tissue, Figure 7 It is the results of immunofluorescence staining of CK13 and CK14 in D3, F3 and A1 cells. From Figure 4 It can be seen that the D3, F3 and A1 cell lines are all typical polygonal stone step-shaped squamous cells, which are the same shape as normal esophageal squamous epithelial cells.

...

Embodiment 3

[0088] Example 3 Organoids

[0089] D3, F3, and A1 cells can all be cultured into organoids, and D3 cells are taken as an example for illustration.

[0090] D3 cells were digested by TrypLE into a single cell suspension, and every 5000 cells were mixed with 55ul Matrigel to form a suspension and added to a 24-well culture plate, and the organoid medium was added after solidification. The fresh organoid medium was replaced every two days, and cultured for 12-15 days, during which the growth of the spheroids was recorded. After the organoid matures, discard the organoid medium, add 500 μl of pre-cooled cell recovery solution, incubate on ice for 45 min~1 h, centrifuge at 1000 g for 5 min, and discard the supernatant. Then add PBS to wash once, discard the PBS washing solution by centrifugation, and add 4% paraformaldehyde solution to fix at room temperature for 1 h. Afterwards, the supernatant was discarded by centrifugation, followed by adding 20% ​​sucrose solution for dehyd...

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Abstract

The invention belongs to the technical field of cell engineering, and particularly discloses a method for constructing a mammalian esophageal squamous epithelial immortalized cell line, the constructed cell line and an organoid cultured by the constructed cell line. The construction method comprises the following steps of: taking primary esophageal squamous epithelial cells as host cells, infecting the host cells by cDNA (hTERT cDNA) retrovirus containing hTERT genes, and screening by selecting a culture medium to obtain the immortalized 'normal' cell line. Meanwhile, the cell line can be cultured into a normal esophageal squamous epithelial organoid, and provides a research basis for researching development and growth of mammalian esophagus, stem cell maintenance, epithelial proliferation and differentiation homeostasis and esophageal diseases, particularly pathogenesis and therapeutic drug research and development of esophageal squamous carcinoma (ESCC).

Description

technical field [0001] The application belongs to the technical field of cell engineering, and more specifically relates to a construction method of an esophageal squamous epithelial immortalized cell line, the constructed esophageal squamous epithelial immortalized cell line and esophageal organoids formed from the cell line. Background technique [0002] Esophageal cancer is a common digestive tract tumor, and about 300,000 people worldwide die from esophageal cancer every year. Esophageal cancer is divided into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), and its incidence and mortality vary greatly from country to country. my country is one of the regions with a high incidence of esophageal cancer in the world, and esophageal squamous cell carcinoma (ESCC) accounts for more than 90%, with an average of 150,000 deaths per year. There are more men than women, and the age of onset is more than 40 years old. The pathogenesis of esophageal ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/54C12N5/10
CPCC12N15/86C12N9/1276C12N5/0602C12Y207/07049C12N2740/10043C12N2800/107C12N2510/04
Inventor 姜伟余茜颖袁徽杨亚男徐蕾
Owner 汉姆德(宁波)智能医疗科技有限公司
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