Fee-for-Service iPSC Line Generation

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Fee-for-Service iPSC Line Generation

 

                                   
Description of Reprogramming Services

 

CCRM performs reprogramming of somatic cell types to induced pluripotent stem cells (iPSCs) for clients in both academia and industry. We specialize in non-integrative strategies and routinely reprogram both fibroblasts and cells isolated from peripheral or cord blood samples. We accept cryopreserved vials of low passage primary fibroblasts, freshly collected blood samples or frozen buffy coat.

 

Reprogramming using alternative methods, or using alternative cell types may be possible. Please contact us to discuss your project further.

 

The Table below describes an example of the Reprogramming and Characterization Services that we provide:

 

Table 1: Reprogramming and Characterization Services Work Plan

 

 

Activity

Description

Stage 1

Parental Cell Expansion, Banking and Mycoplasma Testing

Cryopreserved fibroblasts are thawed. If the culture is mycoplasma negative, cells are expanded to obtain a sufficient number for reprogramming. Likewise, if necessary, target cells are isolated from fresh blood samples and expanded in culture until a sufficient number is obtained for reprogramming.

Stage 2

Transduction of Reprogramming Factors

hOct4, hSox2, hKlf4 and hc-Myc are introduced using Sendai virus or episomal transfection methods or retro/lenti viruses.

Stage 3

Reprogramming Time Course

Seven days following induction, reprogramming cells are re-seeded onto 10-cm dishes. hESC medium is exchanged daily until approximately day 28, until authenticated (identified using in-house established and validated criteria) colonies are ready to be picked.

Stage 4

Picking Authentic Colonies

Authenticated colonies are identified, cleaned and picked into individual feeder-coated wells.

Stage 5

Expansion of Induced Pluripotent Cell Lines

iPSC lines are expanded under the preferred culture conditions to establish five vials of cryopreserved cells for each line.

Stage 6

Characterization of Induced Pluripotent Cell Lines

The following characterization assays will be performed and provided in a report:

  • Karyotyping for two lines per reprogramming experiment. Cell lines with normal karyotypes will be further analyzed
  • Analysis by flow cytometry for OCT4, NANOG, SSEA4, TRA-1-60 and TRA-1-81
  • qRT-PCR for pluripotency markers
  • Cell line authentication (STR analysis) to confirm parental origin
  • Differentiation analysis of two markers for each germ layer

 

Release Criteria

 

All iPSC lines are accompanied by the characterization data specified in Table 2.

 

Table 2: iPSC Characterization

 

Test

Acceptable Release Criteria

Mycoplasma testing 

Negative

Cell morphology by light microscopy

Positive comparison to hESC control 

Karyotype Normal**
Cell line authentication (STR analysis) Must match parental control**

Pluripotency gene expression by RT-qPCR

Oct4

Nanog

 

Rex1

Dnmt3b

 

Normalized-relative gene expression ≥80% of hESC standard

Characterization of differentiation propensity in embryoid bodies Normalized up-regulation relative to undifferentiated cells of same iPSC line and compared with hESC standard

Pluripotency protein expression by flow cytometry

OCT4

TRA-1-60

SSEA4

 

≥80% of cell population express OCT4, TRA-1-60, and SSEA4.
 

Post-thaw viability Undifferentiated colony count 7 days after than ≥10

 

**Assessed by an independent laboratory

 

A final service report will be prepared that includes all of the protocols used, and data collected throughout, iPSC manufacturing.

 

Pricing and Payment Structure

 

The cost to reprogram a sample includes the delivery of two karyotyped and fully characterized iPSC lines. 

 

Every research project is different. Customized projects may have additional costs. CCRM members are eligible to receive a special price. Please contact us to discuss your specific requirements so that we can help you design a successful experiment.

 

Additional Services

 

CCRM also offers the following services on a cost recovery basis:

  • Mycoplasma testing
  • Immunohistochemistry
  • Flow cytometry
  • qPCR
  • Quality analysis and quantification of DNA, RNA 
  • Media testing
  • Embryoid body assays
  • Cell stock expansion and cell banking

 

 

Services Under Development

 

  • iPSC banking and distribution
  • Reporter cell line generation

 

Annual iPSC Training

 

“Thank you for organizing an amazing workshop. I learned all I needed to know about iPS cells.” – 2012 University of Calgary trainee

 

CCRM hosts a Human Induced Pluripotent Stem Cell (HiPSC) Workshop to teach participants how to apply the processes involved in the production and maintenance of iPSCs, and gain a strong foundation in standardized characterization methods to ensure quality stem cell lines are being produced.

 

The workshop is also an excellent opportunity for participants to build a national and international network of colleagues whose interests are in iPSC technology. During the workshop, the following is taught:

 

  • Theory of iPSC production and underlying technical methodologies applied to achieve reprogramming.
  • Hands-on basic cell culture for human ESCs, iPSCs and primary fibroblast lines, including media preparation, colony picking and expansion, cryopreservation and revival.
  • Induction of fibroblasts and blood cells using various transgene delivery methods.
  • Hands-on identification of emergent iPSC colonies and their isolation.
  • Characterization of established iPSCs via immunostaining, quantitative PCR and flow cytometry.
  • Differentiation of iPSCs along desired embryonic lineages.

 

“I wanted to thank you all for putting together the iPSC workshop - it was a great introduction to working with iPSCs, and I enjoyed the hands on practice. The preparatory lecture and your guidance during the lab time was invaluable.” – 2012 workshop participant.

 

If you would like to receive information about CCRM’s next HiPSC workshop, please indicate your interest by e-mailing Emily Titus, Project Manager and Scientist, Cell Reprogramming, emily.titus@ccrm.ca

 

To view a copy of CCRM’s 2013 HiPSC workshop program, please click here.