BSCC core facility

Human induced pluripotent stem cell (hiPSC)-derived disease-specific cell models, coupled with the progress in genome editing technology, serve as an opportunity to study pathophysiological mechanisms in disease-relevant cell models. We provide locally derived human pluripotent stem cell lines (hPSC), genome editing, and generation of hiPSC lines from somatic cells provided by the client. BSCC operates on a fee-for-service basis in providing custom hiPSC derivation and generation of hPSC knockout lines using the latest technologies.

1. Cell and reporter lines distribution

Human embryonic stem cells (hESC) and hiPSC derived locally are provided upon request. We can also provide OCT4 and SOX2 reporter hiPSC lines. Follow this procedure:

• Place request. We can provide several locally derived hESC and hiPSC lines derived from healthy donors. In addition, we can provide OCT4 and SOX2 reporter hiPSC lines. Select the cell line of interest from the table.
• To start the process, contact BSCC Director Timo Otonkoski, or BSCC core manager Ras Trokovic. Human pluripotent stem cells can be delivered live or frozen.
• Sign order form and MTA. To complete your order, we will need to complete an order form and MTA.
• Confirm shipping info. Once the order and MTA forms have been received and accepted you will need to confirm your shipping information and your Courier account number, if necessary.
• Payment. Payment is due once you have received the shipment.

2. Generation of knockout hPSC line

We offer the generation of knockout hPSC lines using CRISPR/ Cas9 technology. For more information on genome editing services please contact BSCC Director Timo Otonkoski or BSCC core manager Ras Trokovic. A signed order form and MTA will be needed. The genome-editing process and time-line are as following:

Design and verify gRNAs. This phase takes about 2 weeks to complete:

• Selection of the cell line that will be edited (hESC or hiPSC)
• Planning of the optimal CRISPR/ Cas9 targeting strategy for the gene of interest
• Design and verification of the screening strategy (primer design and PCR optimization)
• Ordering of 2-4 guide RNAs and validation of their cutting efficiency
• Final report of the plan

CRISPR/Cas9 mediated gene knockout in an hPSC line. This phase takes up to 6 months to complete:

• Expansion and adaptation of the hPSC line to single-cell passaging
• Testing for mycoplasma
• Electroporation of selected RNP into the hPSC
• Single-cell clone establishment of electroporated hPSCs by FACS sorting or limited dilution
• Screening of plates for the hPSC colonies using IncuCyte S3
• Expansion and screening of targeted clones
• Expansion of selected clones and verification of targeted knockout event by PCR and Sanger sequencing
• Testing for mycoplasma and freezing of verified knockout hPSC clones (5 vials per line)
• Final report and delivery of genome-edited hPSCs to the client

Payment is due once you have received the cells

3. Derivation and characterization of hiPSC lines

We offer non-integrative (Sendai virus and episomal vector) technologies for reprogramming somatic cells. The standard cell types used are skin fibroblasts and blood mononuclear cells. Special arrangements can be made for the reprogramming of other cell types. Currently, the method is based on the combination of the four "Yamanaka factors" (OCT4, SOX2, KLF4 and C-MYC). We are using Sendai viruses to reprogram blood cells. We can reprogram fibroblasts using CRISPR-Cas9-based gene activation (CRISPRa) or a conventional transgene reprogramming system. We guarantee the derivation of two hiPSC lines from the cells provided by the client. In the case reprogramming is not successful, there will be no charge for this attempt. To start the service you should:

• Obtain ethical approval for the generation of hiPSC lines. Before hiPSC induction can be initiated from the patient cells, the client needs to obtain ethical approval for the experiment from the local ethics committee. For the users from the Academic Medical Center Helsinki, see instructions (In Finnish).
• Sign order form and service agreement form. Before we start we will need to fill and sign the order form and service agreement for the University of Helsinki or the service agreement for external users.
• Shipment of starting cells. We do not have a possibility to culture mycoplasma positive cells. It is thus important that you test cells for mycoplasma before shipping them to us.
• Reprogramming. Before we start we will consult with you about the technology that we think is the most appropriate for reprogramming your cells. Process guideline and basic characterisation of hiPSC.
• Payment. Payment is due once you have received the cells.

4. Continuous visualization of live cells

The IncuCyte S3 automatically acquires and analyzes images around the clock, providing an information-rich analysis. IncuCyte S3 accommodates up to six microplates at a time. Users can schedule experiments at different image acquisition frequencies and magnifications in parallel. Remote, networked access with unlimited, free licenses. The price is 2€/ hour, this is intended to cover the annual maintenance fees of the equipment.

• First-time users have to register by sending an e-mail to Laboratory Analyst Anni Laitinen
• Registered users can create/ edit your reservations at reservation calender

5. Training
BSCC offers individual customized training for clients. Basic knowledge and experience in aseptic lab work are required. To inquire about the training opportunities contact BSCC Director Timo Otonkoski, or BSCC core manager Ras Trokovic.

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