A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Abstract Background and Aims ADAM17 is a disintegrin and metalloproteinase initially described to cleave the tumor necrosis factor α (TNFα). Currently, it is known that it can also release ectodomains of a diverse variety of molecules such as, transforming growth factor α (TGFα), L-selectin, and angiotensin-converting enzyme 2 (ACE2). It has been shown that ADAM17 protein expression increases in kidney mesangial cells after incubation with high glucose media mimicking what has been observed in diabetic patients and experimental models of diabetic nephropathy. We now studied the effect ADAM17 deletion on human kidney cells (HKC-8) in a 3D spheroids in vitro cell culture incubated with high glucose, low glucose and mannitol medium resembling the in vivo human kidney diabetic environment. Method ADAM17 deletion was performed using the CRISPR/Cas9 technology. HKC8 cells grew inside a RGD-functionalized dextran hydrogel to obtain 3D spheroids. 13 days post-seeding, the spheroids were incubated with 35mM of D-glucose (HG), 5mM of D-glucose (LG) or 35mM of mannitol as osmotic control for 6h, 24h or 72h. The quality of the established 3D cell culture of mature HKC-8 spheroids was assessed by Aquoporin-1 and Glut-1 staining. After incubations quantitative-PCR analyses were performed for fibrotic and inflammatory markers. Immunofluorescence for fibrotic markers was performed on HKC-8 spheroids incubated for 72h. Results High glucose (HG) medium induced CCL5 gene expression on wild-type HKC-8 spheroids after 6h and 24h of incubation in comparison with the control group. Interestingly, in the ADAM17-deleted spheroids, CCL5 gene expression maintained similar to control after 6h of incubation with HG medium and tended to decrease after 24h of incubation in comparison with the wild-type. Collagen IV gene expression was increased in the wild-type spheroids incubated with HG in comparison with the control group. In ADAM17-deleted spheroids, Collagen IV gene expression was significantly decreased in the cells incubated with HG in comparison with the wild-type cells incubated with HG. HG increased the expression of α-SMA, fibronectin and Collagen IV in wild-type spheroids. Adam17 deletion blocked the increase of α-SMA, fibronectin and Collagen IV expression compared with wild-type cells after 72h of incubation. Conclusion ADAM17 blockade protects against fibrosis and inflammation in human kidney tubular spheroids under high glucose.

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In vitro cell culture of patient derived malignant pleural and peritoneal effusions for personalised drug screening

10.21203/rs.2.24824/v2 ◽

2020 ◽

Author(s):

Cheng-Guang Wu ◽

Francesca Chiovaro ◽

Alessandra Curioni ◽

Ruben Casanova ◽

Alex Soltermann

Keyword(s):

Cell Culture ◽

Drug Screening ◽

Drug Testing ◽

3D Cell Culture ◽

Mesothelial Cells ◽

Screening Methods ◽

Hanging Drop ◽

Strong Response ◽

Sensitivity Testing

Abstract Background Malignant serous effusion (MSE) denotes a manifestation of metastatic disease with typical high concentrations of both cancer and immune cells, making them an ideal resource for in vitro cytologic studies. Hence, the aim of the study was to investigate the features of 2D and 3D MSE culture systems as well as their feasibilities for in vitro drug screening. Methods Pleural and peritoneal effusions from 8 patients were collected and processed for 2D monolayer and 3D hanging drop cell culture into GravityPLUS™ plates. Representative markers for cell components, proliferation rate and tumour classification were investigated by immunohistochemistry, followed by absolute quantification using a digitalised image analysis approach. Further, we implemented another 3D cell culture model based on a low attachment method for in vitro drug sensitivity testing of carboplatin, pemetrexed and pembrolizumab for 5 patients. Results Monolayer cell culture was favourable for the growth of mesothelial cells, while hanging drop culture in GravityPLUS™ plates showed better ability for preserving cancer cells, inducing positive diagnostic markers expression and restraining the growth of mesothelial cells. For in vitro drug testing, MSE from five patients presented various drug sensitivities, and one case showed strong response to PD-1 checkpoint inhibition (pembrolizumab). For some patients, the application of combinatorial drugs had better therapeutic responses compared to monotherapy. Conclusions Digitalised quantification of data offers a better understanding of different MSE culture models. More importantly, the proposed platforms are practical and amenable for performing in vitro chemo-/immunotherapeutic drug testing by using routine cytologic MSE in a personalised manner. Next to cell blocks, our work demonstrates the prognostic and predictive value of cytologic effusion samples.

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