standard operating procedure -...

Standard Operating Procedure Number:

Title: Fibroblast Culture from Skin Biopsy for StemBANCC

Approval Date Implemented: 04.2013

Name Signature Date

Author Sally Cowley Sam Evetts [biopsy plating and outgrowth] Jane Vowles [expansion and cryopreservation]

15.04.2013

Principal Investigator Sally Cowley

QA

Change History Reason for Issue/Change Summary

Version No/Date Issued

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1.0 Purpose StemBANCC will recruit 500 patients for creating induced Pluripotent Stem cell lines. Patient recruitment will take place in several centres across Europe. This SOP is to standardise the process by which skin biopsies are plated and fibroblast cultures are established from the biopsies, as this first stage of the procedure can impact upon the efficiency of reprogramming the cells and the quality of the iPS cell lines produced.

2.0 Scope

1) Plating of the skin biopsy 2) Outgrowth of fibroblasts from the biopsy followed by expansion of the fibroblasts over 3 passages 3) Cryopreservation of passage 3 fibroblasts to provide standardised stocks of cells that will then be banked and serve as the starting point for reprogramming to iPS cell lines. 4) Sending frozen vials for reprogramming and banking

3.0 Related Documents

Patient Information Sheet and Consent Form Patient Records StemDB database Related SOPs: SOP. Traceability of derived cell lines for StemBANCC Previous SOPs: SOP. Clinical Procedures: Skin Biopsy SOP. Anonymisation and recording of skin biopsy samples for StemBANCC Next SOPs: Newcastle SOP iPS reprogramming protocol

4.0 Responsibility All labs processing received biopsies for StemBANCC have responsibility for implementing this SOP and suggesting amendments to the co-ordinating authors (Sally Cowley UOXF).

5.0 Health Safety & Environment Biopsies and derived fibroblasts are to be handled within a Class II safety cabinet to protect the worker from possible adventitious agents. The patient should have been tested and be negative for HIV, HBV and HCV, but pathogen screening will never be comprehensive. Local, national and EU health and safety regulations must be adhered to.

6.0 Materials & Equipment

Product name Supplier Catalogue number

Advanced DMEM Life Technologies 12491

Fetal Bovine Serum USDA approved (for future import of

PAA (suggested) A15-304

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cell lines)

Gluta-MAX 100X Life Technologies 35050

Antibiotic-Antimycotic, 100X Life Technologies 15240-062

Penicillin-Streptomycin 100x PAA P11-010

DMSO Sigma D2650

Trypsin/EDTA 0.05%/0.02% in PBS PAA L11-004

TrypLE Express Life Technologies 12604-013

PBS w/o Ca &Mg PAA H15-002

MycoAlert mycoplasma detection kit Lonza LT07-118

MycoAlert™ positive control Lonza LT07-518

100mm sterile plastic plate for dissecting biopsy (doesn’t have to be tissue culture treated)

any

6-well tissue culture plate Corning 3516

T25 tissue culture flasks, vented caps Corning 3056

T75 tissue culture flasks, vented caps Corning 430725

Cryotube 1.8ml, external threaded Nunc 375418

‘Mr Frosty’ controlled rate freezing tub Nalgene 479-3200

isopropanol

Labels for cryotubes

NB Advanced DMEM is preferable to DMEM, as DMEM is buffered for use in 10% CO2 ADMEM Lot. No._______________________

USDA approved FBS Lot. No. (recommend testing several batches of FBS for fibroblast growth, then using one batch for a year, frozen in aliquots at -20) __________________ Other Equipment Surgical scalpel no.22 (sterilised) Forceps (sterilised) Glass Coverslips, sterilised (22mm is best) 37°C water bath Fibroblast medium 500ml Advanced DMEM 56ml FCS 5.6ml Gluta-MAX 100X 5.6ml antibiotic-antimycotic 100x (for plating biopsy) Or 5.6ml penicillin/streptomycin 100x (for fibroblast expansion) Mix well, Store 4°C for up to 4 weeks or freeze in single-thaw aliquots Mix well after thawing 2X freezing Medium 22ml Fibroblast medium

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10ml FCS 8ml DMSO Mix well, Store -20°C in suitable aliquots Mix well after thawing

7.0 Procedure NOTE 1: All procedures to be carried out in a Class II safety cabinet NOTE 2: Incubations are performed in a humidified 37 degree C, 5% CO2 incubator NOTE 3: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. For processing the biopsy, surgical equipment should preferably be autoclaved. It may alternatively be sterilized by soaking in 70% ethanol for 20 minutes; however, in this case it is important to air-dry in the hood, or rinse in sterile PBS before use since ethanol is toxic to the tissue. NOTE 4: Work with one sample at time to avoid possible labelling errors. 7.0.1 Record biopsy information

According to SOP Traceability of derived cell lines for StemBANCC 7.1 Establishment of Fibroblast Cultures 7.1.1 Prepare skin sample

Wash skin samples in PBS by gently shaking or agitating in a 50-ml polypropylene centrifuge tube.

For relatively large skin samples (e.g., foreskin, surgically removed samples, or cadaver skin): Place sample on the lid of a 100-mm tissue culture dish and spread it out with the epidermal side down. Remove the subcutaneous tissue by scraping the dermal side using two pairs of eye forceps. Cut skin into strips of ∼0.5-cm width using a surgical scalpel.

Alternatively, For smaller skin samples (e.g., punch-biopsy samples): Place the sample on a 100-mm tissue culture dish and excise the subcutaneous tissue using a surgical scalpel and a pair of forceps.

7.1.2 Culture fibroblasts

Place the dermal sample on the lid of a 100-mm tissue culture dish and cut it into small (2- to 3-mm) squares. Fibroblast outgrowth occurs only from sharply cut edges. Thus, it is crucial to use a new, fine surgical scalpel; disposable surgical blades (no. 22) can be used for this purpose.

Label a 6-well plate with date, line code, operator’s initials and passage number (p0), ensuring the bottom of the plate is also labelled.

Place ~4-5 skin pieces in the centre of a well of the 6-well plate.

Place a sterile 22-mm glass coverslip gently over the skin specimens (Fig. 1).

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Skin specimens need to be attached physically to the plate. This can be achieved most effectively by making a sandwich using a coverslip (Fig. 1). The coverslip also assists the growth of fibroblasts by maintaining the microenvironment.

Add a few drops of fibroblast medium into the space below the coverslip (by applying at the edge of the coverslip so that it is drawn under), then add 2 ml of medium to the dish or well, gently so as to avoid disturbing the skin specimens.

Place the culture in a humidified 37 deg C, 5% CO2 incubator, positioned so that it will not be disturbed for the first few days. Check the fibroblast outgrowth every 3 to 4 days under an inverted phase-contrast microscope and change medium every 3 to 4 days, taking care not to agitate the coverslip. Initial outgrowth should be detectable within 3 to 4 days (Fig. 2). The coverslip should be left on the skin specimens until the culture becomes confluent.

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7.2 Expansion of fibroblasts 7.2.1 Split cells into a T25 flask When the fibroblasts are fully confluent around the biopsy and spreading out they should be split. See figure 2.

Equilibrate a T25 flask with 4ml Fibroblast medium @ 37°C & 5% CO₂ for 15 minutes minimum.

Warm Trypsin/EDTA or TrypLE to 37°C.

Aspirate medium from biopsy, wash with PBS taking care to wash under coverslip, aspirate. Fibroblasts sometimes migrate over the surface of the coverslip instead of the tissue culture plate. If this is the case (as judged by focusing up and down with the microscope) transfer the coverslip into a new dish, wash it with PBS as in this step, then harvest cells by trypsin treatment.

Add 1ml warmed Trypsin/EDTA or TrypLE, swirl to cover biopsy then remove 0.5ml.

Place @ 37°C for 5 minutes.

Check under the microscope that the fibroblasts have rounded up but not the other cell types eg keratinocytes. If not return to incubator for 1-2 minutes and check again. Tap plate to loosen cells.

Using P1000 Gilson pipette, add 1ml Fibroblast medium, washing off cells and paying particular attention to under coverslip. Transfer to equilibrated T25 flask.

Repeat with a second 1ml of medium.

Check under the microscope that most of the fibroblasts have been removed but not the biopsy and surrounding keratinocytes.

Add 2ml fibroblast growth medium to biopsy and return to incubator to continue culture as a back-up in case needed. When this backup culture is approaching confluence, freeze cells in 1 vial (as p2) as a back-up frozen stock, in case of failure of the final 6 vial stock for any reason

Label T25 with date, line code, operators initials and passage number (p1)

Return to incubator.

Replace medium every 2-3 days (5ml)

During the first medium change take 2ml of the ‘spent’ medium from the fibroblast culture and test it for mycoplasma according to the mycoplasma kit instructions. If the result is negative, then record this in the StemBANCC database, with date, laboratory site and passage number.

If mycoplasma test is positive, it is highly advised to discard the biopsy, the derived fibroblasts, the associated bottle of medium and thoroughly disinfect hood etc

If there is no chance of obtaining a repeat biopsy, and the sample is declared to be so precious as to not be disposed of (e.g. an extremely rare variant) then there may be justification to quarantine the biopsy, the derived fibroblasts and the medium and treat to remove all traces of mycoplasma. If

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this course of action is pursued, the cells must test negative for mycoplasma at least twice before continuing in the pipeline, the database must have the information recorded, and recipients of the cells must be fully alerted to the history in advance.

7.2.2 Split cells into a T75 flask When fibroblasts are 90% confluent they should be split. See figure 3.

Equilibrate a T75 with 10ml Fibroblast medium @ 37°C & 5%CO₂ for 15 minutes minimum.

Warm TryplE to 37°C.

Aspirate medium from T25 and wash with PBS, aspirate.

Add 1ml TryplE and swirl to cover cells.


Check under the microscope that the fibroblasts have rounded up. If not return to incubator for 1-2 minutes and check again. Tap flask to loosen cells.

Add 2ml fibroblast growth medium to T25, washing off the cells and transfer to the equilibrated T75.

Repeat with a second 2ml and add to same flask.

Check flask under microscope to ensure all cell have been transferred.

Label T75 with date, line code, operators initials and passage number (p2)

Return to incubator.

Replace medium every 2-3 days (13ml) 7.3 Freezing down cells When fibroblasts are 90% confluent in the T75 flask (p2) they should be frozen down.

Cool ‘Mr Frosty’ to 4°C.

Cool freezing medium on ice.

Label 6 cryovials according to SOP. Traceability of derived cell lines for StemBANCCand cool to 4°C in Nunc rack. The passage number on the vial is one more than the flask being harvested from. 1/6 of a 90% T75 contains 0.25-0.5 x 106 fibroblasts, which will provide enough cells upon thawing for subsequent reprogramming (UNEW SOP 10)

Warm TryplE express to 37°C.

Aspirate medium from the T75 and wash with PBS, aspirate.

Add 2ml TryplE Express and swirl to cover cells, then immediately remove 1ml.


Check under the microscope that the fibroblasts have rounded up. If not return to incubator for 1-2 minutes and check again. Tap flask to loosen cells and check all are detached.

Add 2ml Fibroblast medium to T75.

Working quickly add 3ml ice cold 2x freezing medium, mix well.

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Aliquot 1ml/cryovial.

Place vials in Mr Frosty and place in -80°C freezer overnight. This system ensures controlled-rate freezing at -1 degC/minute.

Transfer to vapour phase liquid nitrogen storage the next day. The cells deteriorate rapidly at -80, so be well-disciplined about transferring to nitrogen storage, and do not leave vials in the -80°C freezer for longer than 5 days. Ensure temperature is not allowed to rise during transit to nitrogen storage – keep Mr Frosty embedded in dry ice (frozen carbon dioxide pellets).

Complete logbook/computer records for cryostorage facility + StemDB submission according to SOP Traceability of derived cell lines for StemBANCC

7.4 Sending frozen vials of fibroblasts

Retain 2 vials as a backup

Arrange for 2 frozen vials to be sent to Reprogramming centre: o UOXF for Parkinson’s Disease patient samples:

Dr Sally Cowley Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE UK +44 (0) 1865 275545 [email protected]

o UNEW for all other samples:

Dr Lyle Armstrong Institute of Human Genetics University of Newcastle upon Tyne International Centre for Life Central Parkway Newcastle upon Tyne NE1 3BZ UK Tel: +44 (0) 191 241 8695 [email protected]

Arrange to send 2 frozen vials to Birmingham Biobank: o Dr Jane Steele

Human Biomaterials Resource Centre

College of Medical and Dental Sciences University of Birmingham Edgbaston Birmingham B15 2TT UK

Tel: +44 (0) 121 414 7668 [email protected]

mailto:[email protected]

mailto:[email protected]

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Ensure the vials are packed in a leak-proof biohazard-grade plastic bag or vessel, with sufficient absorbent material to absorb all the liquid in cryovials in case of mechanical failure and thaw. Temperature fluctuation of the Cryovials must be minimised throughout the packaging process – they should be kept under dry ice at all stages of removal from nitrogen storage and packaging.

Ensure sufficient dry ice (10 kg) is included on top of the vials in the package to last for several days, in case of delay during transit, in an expanded polystyrene box.

Check with the person receiving the package in advance, to ensure that it will be expected and received Send vials at the start of the week to avoid weekend delivery, or delay over the weekend. Check in advance about national holidays, departmental closures over Christmas etc.

Send package by overnight delivery. FedEx is recommended. Charge to your StemBANCC grant. To minimise transport costs, send cells in batches, unless urgent.

Include sender contact details and details of contents: Clinical samples for medical research use only Value for customs £5

Complete StemBANCC Material Transfer Form and send copy with samples for Recipient to sign and send to project office

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Figure 1 Skin explant culture a) top view; b) side view

Figure 2. Typical appearance of fibroblasts cultured from skin biopsy.

Figure 3 Fibroblasts ready for passaging

Skin biopsy

Keratinocyte

outgrowth

Fibroblast

outgrowth

Fibroblasts ready for passaging

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Figure 4. Trypsinising the fibroblasts

Figure 5. Fibroblast confluency after splitting

Expected minimum confluency 48

hours after splitting

Fibroblasts rounded up,

Biopsy still attached

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Figure 6. Fibroblasts ready for splitting

8.0 Troubleshooting

9.0 Appendices

9.1 If cells need to be thawed from liquid nitrogen storage

Equilibrate a T75 with 15ml Fibroblast medium @ 37°C & 5%CO₂ for 15 minutes minimum.

Bring cells from liquid nitrogen store on dry ice.

Thaw cells by gently swirling vial in 37°C water bath until just a small ½ pea size of ice is left. Take care not to get water on cap.

Wipe vial dry and wipe with alcohol dampened tissue.

Transfer vial to hood and transfer entire contents into equilibrated a T75.

Label T25 with date, line code, operator’s initials and passage number from vial. The passage number was already increased by 1 on the frozen vial

Rock flask to mix and place carefully in incubator where it will not be moved.

Replace medium after 12-16 hours to remove residual DMSO.

Replace medium every 2-3 days (15ml).

Cells ready for splitting/freezing, 90% confluent

100µm