More than 5 years of experience with Isolation, Expansion and Immortalization of human cells from biopsies

SIRION BIOTECH over the years has generated various immortalized human cell lines displaying high functionality in cell based assays; comparable to parental primary cells. Examples for immortalized cell lines include human taste bud-derived cell lines, human umbilical vein endothelial cells (HUVEC), and human bronchial epithelial cells (NHBE).

 

Examples:

Figure 1: Tube formation of a conditionally immortalized HUVEC cell clone expressing large t (ts) and hTERT expanded for 20 passages (left), and expressing VE Cadherin (center). Morphology of a human taste bud-derived cell line (right).

Expanding primary cells via the expression of cell cycle regulating genes

  • Cell cycle regulating genes are introduced into primary cells that have retained a proliferative capacity. This is done via lentivirus-mediated cell transduction.
  • Towards this purpose, SIRION BIOTECH has designed an array of lentivirus vectors expressing each a single or a combination of cell cycle regulating genes. 
  • In a next step, lentivirus-transduced cells with stable integration of the transgene are selected for stable gene expression by antibiotic selection
  • Towards this purpose, SIRION BIOTECH optimizes cell culture conditions for primary cells and conditions for viral gene transfer.

A wide variety of cell types were immortalized by applying SIRION BIOTECH's immortalizing genes and combinations. Some of these combinations go beyond what is industry standard and therefore are proprietary to SIRION BIOTECH. For example, some of the lentiviruses expressing shRNAs knocking down genes. Or take certain viral vectors comprising both AV and LV instead that allow for expansion of primary cells prior to stable gene transfer using lentivirus vectors. This is of specific value for cell types with poor replicative competence. 

 

Array of lentiviruses used:

For generation of stable cell pools a series of proprietary lentivirus vectors are used alone or in combination; please inquire for further details. The resulting cell pools stably expressing the cell cycle-regulating gene are characterized for cell morphology, viability and optionally for functional parameters. 


Clonal cell line generation and cell banking

Of the cell pools that reliably express  cell cycle regulating genes, 

  • clonal cell lines are established by means of limiting dilution and seeding in 96-well plates. 
  • The clonality of each cell line is verified by back tracking to single cells using the Clone Select Imager (Genetix)
  • Each cell clone is expanded to 1x10E7 cells and cell banked and a master cell bank established (MCB)
  • Each cell clone from the MCB is further expanded and cultured for a minimum of 20 passages, corresponding to >40 population doublings
  • good cell clones are functionally characterized after 20 passages and possibly expanded further 

The concept of conditional immortalization

Cell cycle exit is required for terminal differentiation of primary cells. 

  • The issue: continuous expression of cell cycle regulating genes may interfere here, making it desirable to shut off expression of a gene at the point of differentiation
  • The thermosensitive variant of the large T antigen (large T (ts)) is comparable to a switch, having the large T antigen expressed as active protein at 37°C, which becomes inactive at 37°C.
  • The largeT (ts) mutant, though, is still being used for immortalization of a large number of human and rodent cell lines [1]
  • In combination with genes capable to activate the human telomerase gene, including hTERT, expression of large T (ts) allows for generation of immortalized cell clones.

However, not all cell types can be successfully immortalized by this approach. SIRION BIOTECH expands the concept of conditional immortalization in case other cell cycle-regulating genes have to be used for immortalization.

  • SIRION BIOTECH would regulate the expression of these genes with TET-inducible gene expression that it is offering as tools for research and development
  • Towards this purpose, SIRION BIOTECH has developed a range of inducible lentivirus gene expression vectors that are used to generate stable cell clones
  • The cell clones are expanded in the presence of Doxycyclin “TET-on” and express the cell cycle regulating genes in the presence of the antibiotic
  • For terminal differentiation, cells are cultured without Doxycyclin, turning off the gene expression and facilitating cell cycle exit.   

 [i] Parmjit S et al. PNAS 1991, 88:5096-5100.

 

How to work with SIRION BIOTECH

Please refer to our page "How to work with SIRION BIOTECH" to learn more about how working with us looks like.