Rapid and Non-Enzymatic In Vitro Retrieval of Tumour Cells from Surgical Specimens

Abstract

The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm3 cubes termed explants, which are cultured 1–3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines.

Spheroid-based 3D Cell Cultures Enable Personalized Therapy Testing and Drug Discovery in Head and Neck Cancer

“…With our new intermediate cell line PiCa from our group, we were able to conduct treatment experiments at a larger scale and confirmed current therapy expectations (23). While this might seem like a circumstantial fact, it supports our theory that PiCa cells reflect actual human tumor behavior better than other, more aged and passaged cell lines that underwent numerous passages.…”

“…The UPCI-SCC-154 cell line from oropharyngeal HPV-associated cancer was obtained by U. Schoetz (Department of Radiation Oncology, Munich, Germany). The intermediate cell line PiCa originated from our associated workgroup at Großhadern campus (23). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Biochrom, Berlin, Germany) supplemented with 10% fetal bovine serum (Gibco Life Technologies, Paisley, UK), 1% penicillin/streptomycin (Biochrom), 1% sodium pyruvate (Biochrom), 1% non-essential amino acids (Biochrom) and 1% L-Glutamine (Gibco Life Technologies).…”

“…None of the techniques copies the variability in terms of genetic alterations and cell heterogeneity in the same way as the real cancer does as they often comprise the use of heavily altered and aged cell lines with few reliable matching characteristics to actual human tumor cells. To address this problem, we were recently able to generate a new cell line referred to as "PiCa" derived from fresh human cancer biopsies without enzymatic degradation and with tumor-forming capacity in animal models (23). With limited passages and characterization confirming conserved cellular markers on the cell surface, on the road to future trials with primary human cells, this cell line was our main focus in the development of the following model.…”

Evaluation of innate and adaptive immunity contributing to the antitumor effects of PD1 blockade in an orthotopic murine model of pancreatic cancer

“…Cell culture and luciferase transduction KPC cells were established from spontaneous KPC tumors that were finely minced using scalpels and directly plated (outgrowth method), without mechanical or enzymatic dissociation as previously described. 41 KPC tumors were obtained from crossing the LSL-Kras-G12D with LSL-p53-R172H and PDX1cre mice (NCI-Frederick) and further backcrossing for >10 times to attain animals with a predominantly C57BL6 background. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/mL of penicillin/streptomycin (Gibco) at 37 C, 5% CO 2 in a humidified incubator.…”

Precision Medicine in Oncology: In Vitro Drug Sensitivity and Resistance Test (DSRT) for Selection of Personalized Anticancer Therapy

“…[98] Hagemann et al studied formation of spheroids from the PiCa cell line, which was generated from primary carcinoma specimens without enzymatic digestions of single cells. [99,100] Spheroids were grown using two methods-1) the method of hanging droplet, where cell aggregation is promoted by gravitational accumulation of cells in the water-air interface of droplets; and 2) ultra-low attachment plates (ULA), where cells are prevented from attaching to the surface, which leads to cell aggregation. The authors successfully formed spheroids using both methods and demonstrated reduction of spheroid diameter by treating them with cisplatin, 5-fluorouracil (5-FU), or radiation.…”