The protocols in this section are the Standard Operating Procedures (SOPs) used for the production and testing of cell lines distributed by the NSCB (National Stem Cell Bank). Many of these SOPs may differ from the protocols WiCell teaches in their training courses due to the larger sized lots of hES cells distributed by the NSCB. The Basic NSCB hES Cell Culture Protocols(pdf) are written for researchers new to the field of hES cell culture, while the NSCB SOPs document the methods used to prepare the hES cells for the bank. You will need Adobe Acrobat Reader to access these pdf files.

hES Cell SOPs:

Preparation of hES Cell Culture Medium (SOP-CC-001C)

Thawing hES cells onto MEF feeder layers (SOP-CC-020C)

Medium exchange for hES cells (SOP-CC-022C)

Preparation of Collagenase Solution (SOP-CC-011A)

Pooling of hES cells during passaging (SOP-CC-015A)

Expansion of hES cells on MEF feeder layers (SOP-CC-030C)

Alternative spliting of hES cells on MEFs (SOP-CC-037A)

Selection and Maintenance of hES cells (SOP-CC-025A)

Overfeeding hES cells (SOP-CC-027A)

Cryopreservation of hES Cells for a Large Scale hES Cell Lot (SOP-CC-035D)

MEF SOP's:

Preparation of MEF Medium (SOP-CC-003B)

Derivation of new MEF cells (SOP-CC-006D)

Cryopreservation of MEF cells (SOP-CC-031D)

Thawing MEF vials (SOP-CC-021A)

Passaging MEF cells (SOP-CC-009A)

Propagation and distribution of MEF cells (SOP-CC-018D)

Plating inactivated MEF cells (SOP-CC-013A)

Cryopreservation of inactivated MEF cells (SOP-CC-023A)

Thawing inactivated MEF cells (SOP-CC-024A)

MEF Lot Qualification (SOP-QU-013A)

Cytogenetics SOPs:

Harvest of hES cells for chromosome analysis (SOP-CH-001C)

Preparation of metaphase chromosome spreads from hES cells for cytogenetic analysis (SOP-CH-002B)

G-T-L banding for chromosome analysis (SOP-CH-003C)

Flow Cytometry and Growth Curve SOPs:

Harvesting hES cells for flow cytometry (SOP-CH-101C)

Internal staining of hES cells for flow cytometry (SOP-CH-102C)

Embryonic Stem Cell Assessment by Flow Cytometry-Directly Conjugated Antibodies (SOP-CH-103B)

Growth Characteristics (SOP-CH-104B)

Embryonic Stem Cell Assessment by Flow Cytometry-Indirectly Conjugated Antibodies (SOP-CH-105B)

Staining MEF co-cultured cells for the Growth Curve Assay (SOP-CH-106C)

Fixing, Staining, and Counting hES Cells on MEFs (SOP-CH-108A)

Differentiation SOPs:

Cardiac Differentiation from Human ES Cells – EB Method (SOP-CH-203C)

Generation of Pancreatic Progenitors from hES Cells (SOP-CH-204B)

Immunohistochemistry for Pancreatic Lineage Markers (SOP-CH-205B)

QPCR to assess pancreatic lineage markers (SOP-CH-206C)

Neural Differentiation of hES cells (SOP-CH-207A)

Generation of Hematopoietic Cells from Human Embryonic Stem Cells (SOP-CH-208C)

Cardiac Differentiation Potential and Time Course in EBs: Percentage of Contracting EBs (SOP-CH-209C)

Immunolabeling of Cardiac Markers in EBs (SOP-CH-210C)

Quantitative RT-PCR Analysis of Cardiac Markers to Assess Cardiomyocytes Differentiation from hES Cells (SOP-CH-211D)

Flow Cytometry Analysis of Cardiac Markers to Assess Cardiomyocytes Differentiation from hES Cells (SOP-CH-212B)

Teratoma Initiation in hES Cells (SOP-CH-213A) 

TESTING SOPs:

DNA Testing SOP's:

Extraction of DNA from hES Cells (SOP-CH-014A)

G-Banding Analysis and Image Capture (SOP-CH-009A)

Submission Procedures and Requirements for Short Tandem Repeat (STR) and/or Human Leukocyte Antigen (HLA) Testing (SOP-SS-006B)

RNA Testing SOP's:

Extraction of RNA from hES cells (SOP-SS-025D)

Expression Chip Data Analysis (SOP-CH-311B)

gDNA preparation and AminoAllyl labeling for Gene Expression (SOP-CH-321B)

RNA preparation and AminoAllyl labeling for Gene Expression (SOP-CH-322B)

Hybridization Using Formamide-based Hybridization Buffer on Maui Station for Gene Expression (SOP-CH-333B)

Sterility & Mycoplasma Testing SOP's:

Apptec_Sterility

Apptec_Sterility B/F

Apptec_Mycoplasma Detection 

Apptec_Mycoplasma + Mycoplasmastasis

Mycoplasma testing by Bionique

Pathogen/Virus Testing SOPs:

Apptec_Co-Cultivation

Apptec_Retrovirus (TSEM) 

Apptec_In vitro Adventitious Agent testing

BioReliance_Bovine Pathogens

BioReliance_Porcine Pathogens

BioReliance_MAP

BioReliance_Test for Inapparent Viruses

BioReliance_HIV 1&2 by PCR

BioReliance_HTLV 1&2 by PCR

BioReliance_HBV by PCR

BioReliance_HCV by PCR

BioReliance_EBV by PCR

BioReliance_HHV-6 by PCR

BioReliance_HHV-8 by PCR

BioReliance_HP B19 by PCR

Thawing SOP's:

Post-Thaw viable cell recovery (SOP-CH-305C)