Tumor cell lines

v  A tumor is an abnormal mass of cells.

v  Benign vs. Malignant

v  Tumors can either be benign (non-cancerous) or malignant (cancerous). Benign tumors grow locally and do not spread. Malignant tumors have the ability to spread and invade other tissues. This process, which is a key feature of cancer, is known as metastasis.

Isolation of tumor cell

v  Successful isolation of tumor cells is critically dependent upon the method to disrupt the extracellular matrix, which consists of a complex mixture of cohesive materials such as connective tissues, glycoproteins, and tissue specific proteins.

v  WHY???

v  Tumor cell are isolated to in vitro culture to generate primary tumor cell lines. There are several methods to isolate primary tumor cells from tissues such as 3D culture, cancer stem cell enrichment and sandwich culture. These primary tumor cells undergo expansion within 7–10 days.

Tumor cell culture

v  Primary tumor cell culture?

·         Sampling of tumor cells.

·         Initial establishment.

·         Disaggregation.

v  Cancer cell lines?

v  Characterization of tumor cell lines.

v  Common problems with tissue cell culture.

Primary Tumor Cell Culture

·         Sampling of tissue cells.

·         Cell bank

v  World federation for culture-having 469 centre in world level.

·         Isolation of tumors from animals.

·         Initial establishment

Transfer of fragment to hood.

Viable cells (remove necrotic factor).

PBS wash (to remove blood).

Disaggregation

·         Mechanical method

v  Scalpels

v  Pulverize

v  Avoid cluster of cells

v  Centrifuge

v  Inoculate

·         Enzymatical method

v  Trypsin

v  Collagenase

v  Elastase

v  Dispase

v  Hyaluronidase

Papin

Culture and Maintenance

Several techniques are available to grow primary cell cultures from tumors; however, very few methods have been found to be promising.

1. Two-dimensional (2D) monolayer cultures
2. Explant-cell culture system
3. Precision-cut slice cultures
4. Three-dimensional (3D) cell cultures
5. Partial enzymatic degradation of stromal cells
6. Sandwich cultures
7. Cancer stem cells isolation approach.

Method to derive Primary Tumor Cell Lines(Explant cell culture)

⇩

This explant cell culture method is suitable for developing “primary tumor cell lines.”

Precision-cut slice cultures:

•      It is similar to the explant-cell culture system however the size of an individual slice is restricted to a specific thickness .

•      160 μm is optimal for growing cells and preserving their morphology.

•      If slices are thinner (e.g. 100 μm), cells might disintegrate and possibility of getting damaged nuclei.

•      By contrast, thicker slices cause difficulties in supplying nutrients and oxygen to cells, lessening their viability.

Method

Chemical reprogramming for tumor cell cultures

•      Recombinant growth factors

•      Hormones are essential to maintain proliferative primary tumor cell cultures.

•      Chemical treatment (Rho Kinase inhibitor Y-27632) can be used on fibroblast feeder cells to proliferate both normal and tumor cells.

•      It is used when above supplements are not enough to maintain the viability of neoplastic cells. i.e tumor cells

Tissue Specific culture media

Importance of Tumor cell lines

•      Compared to normal cells, tumor cells have undergone an array of genetic alterations.

•      These changes underlie cancer development, progression, and drug resistance.

•      Tumor-derived cell lines used to study the biological processes in cancer, as well as screening platforms for discovering and evaluating the efficacy of anti-cancer therapeutics.

•      Cell lines offer the advantages of being easily grown, relatively inexpensive, and amenable to high-throughput testing of therapeutic agents.