Establishment and optimization of co-culture technology for breast cancer organoids

doi:10.3969/j.issn.1674-8115.2021.08.004

Abstract

Abstract: Objective

·To improve the cultivating and passaging method of breast cancer organoids, and establish a co-culture system enriching cancer associated fibroblasts (CAFs).

Methods

·Different types of collagenases (type Ⅰ, type Ⅲ and type Ⅳ) were used to digest fresh tissues from 5 breast cancer patients. The number of cells after tissue digestion was counted by cell counting method, and cell viability was analyzed by cell flow cytometry. Three-dimensional culture of primary breast cancer single cells was carried out by using culture system containing different contents of fibroblast growth factor 7 (FGF7), FGF10 and epidermal growth factor (EGF). The success rate of cell culture and the growing status of organoids were observed and compared. Different centrifugation speeds were used to compare the advantages and disadvantages of passaging methods and simplify the passaging steps. CCK8 assay was used to study the effect of CAFs on the growth of organoids in the co-culture system of primary CAFs and organoids, and the morphological changes of organoids were observed under optical microscope.

Results

·Compared with type Ⅰ and type Ⅲ collagenase, type Ⅳ collagenase got the highest cell yields (P=0.045, P=0.017), and maintained the highest cell viability (P=0.005, P=0.048). By optimizing the composition of organoid medium (omitting FGF7 and FGF10, reducing EGF concentration) and passaging process (improving centrifugal velocity to 900×g), a more economical, effective and rapid method of organoid culture was obtained. Compared with organoids cultured alone, the growth rate (P<0.05) and heterogeneity of organoids increased when organoids were co-cultured with CAFs.

Conclusion

·The optimized culture system can significantly increase the success rate of organoids, simplify the culture steps and reduce the culture cost. The establishment of primary CAFs and organoids co-culture system provides a good in vitro model for the study of breast cancer heterogeneity and tumor microenvironment.

Key words: breast cancer, organoid, improved culture condition, cancer-associated fibroblast (CAF), co-culture

CLC Number:

R73-3

Tian-hao ZHOU, Zhao-chen XIN, Shao-qian DU, Yuan CAO, Jing-xuan XU, Zeng-hong LAO, Hong-xia WANG. Establishment and optimization of co-culture technology for breast cancer organoids[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(8): 1017-1024.

Figures/Tables 4

Fig 1 Comparison of digestion efficiency and cell damage of different types of collagenase in breast cancer tissues

Fig 2 Optimization and improvement of PDO culture and passage conditions

Fig 3 Molecular expression and histological characteristics of PDO

Fig 4 Construction of CAFs and PDO co-culture system and influence of CAFs on morphological heterogeneity and growth rate of PDO

TrendMD

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