University of Pennsylvania
Translational Research Program Laboratory
538-545 BRB 2/3, 421 Curie Boulevard
Philadelphia, PA 19104-6160 / Page 1 of 5
Effective Date: June 7, 2007

Document Number: TRP211-02

Title: Preparation of 293T Culture Medium

Signature/Approval Date:

Originator:Richard Carroll

Reviewed by:Kathleen Haines

1.0 / Purpose
1.1 / This protocol describes the process by which the medium used for routine culture of 293T cells is prepared. 293T cells are used for production of lentiviral vectors.
2.0 / Scope
2.1 / 293T Culture medium is used for routine culture and maintenance of 293T cells (see SOP TRP340).
3.0 / Definitions and Abbreviations
3.1 / TRP- Translational Research Program
3.2 / SOP- Standard Operating Procedure
3.3 / RPMI 1640- A commonly used basal medium
3.4 / FCS- Fetal Calf Serum
3.5 / HI&SF- Heat-inactivated and Sterile Filtered
3.6 / Gln- Glutamine
3.7 / Pen/Strep- Penicillin and Streptomycin
3.8 / HEPES – Buffer used to control media pH
3.9 / IU- International Unit
3.10 / Exp.- Expiration
3.11 / Cat. – Catalog
3.12 / Mfr.- Manufacturer
3.13 / BSC- Biosafety cabinet
4.0 / Responsibility
4.1 / The TRPLaboratory staff is responsible for following this SOP to prepare 293T culture medium.
4.2 / The Laboratory director or his designee will review the records for accuracy.
4.3 / The TRPLaboratory staff will archive the records in a binder entitled: “293T Medium Batch Logs”.
5.0 / Equipment and Materials (use listed or equivalent)
5.1 / Reagents and Materials (use listed or equivalent)
5.1.1 / RPMI-1640with Glutamax and 25 mM HEPES (Invitrogen Cat. #72400)
5.1.2 / FCS, non heat-inactivated (Hyclone Cat. #SH30071.03)
5.1.3 / Penicillin 10,000 IU/ml/ Streptomycin 10,000 ug/ml, [Mediatech/Cellgro Cat. # 30-002-C1]
5.1.4 / 70% Ethanol
5.1.5 / Pipettes, various sizes
5.1.6 / Aluminum foil
5.1.7 / #5262 Labels (Office Depot Cat. #916486)
5.1.8 / Millipore Stericup Filter, 0.22 uM, 500 ml (Fisher Cat. # SCGP U05RE), or
5.1.9 / Millipore Stericup Filter, 0.22 uM, 1000 ml (Fisher Cat. # SCGP U11RE)
5.2 / Equipment
5.2.1 / 37oC waterbath
5.2.2 / Biosafety Cabinet
5.3 / Documents
5.3.1 / Form 211-A: 293T Culture Medium Batch Log
6.0 / Procedures
6.1 / Remove the following items from the refrigerator and allow to warm to room temperature while wrapped in aluminum foil:
6.1.1 / RPMI 1640 containing Glutamax and HEPES (fresh bottle)
6.1.1.1 / If using RPMI 1640 lacking Glutamax or HEPES, these components must be added separately. See Attachment 10.1 for details.
6.1.2 / Pen/Strep 10,000 IU/ml and 10,000 ug/ml, respectively
6.1.3 / FCS, non-heat-inactivated
6.2 / Verify that the Pen/Strep has been stored at 4oC for less than two months.
6.3 / If necessary, thaw bottles of any of the above reagents in a 37oC waterbath. Mark date of thaw on bottle. Leave at 37oC for the minimal amount of time required for thawing.
6.3.1 / The exception to this is FCS. FCS must be thawed in accordance with SOP TRP135.
6.4 / Mix all thawed material by gently pipetting or swirling.
6.4.1 / If mixing by swirling, it is important not to let any of the liquid reach the mouth of the bottle, to minimize contamination.
6.5 / Wipe all warmed reagents with 70% EtOh and bring into BSC.
6.6 / Remove RPMI from a new bottle and discard a portion, as outlined in Attachment 10.1.
6.7 / Combine all components as described in Attachment 10.1.
6.8 / Label bottles as follows:
6.8.1 / 293T Culture Medium
6.8.2 / Batch # (see step 8.3)
6.8.3 / Preparation date
6.8.4 / Expiration date (1 month after preparation or earliest expiration date of any component, whichever comes first).
6.8.5 / Initials of preparer
6.9 / If desired, the medium can be sterile-filtered through a 0.22 uM Stericup filter. However, this is not required.
6.10 / Store in the dark at 2-8oC.
6.11 / Complete form 211-A.
6.12 / After review, file Form 211-A in the 293T Medium Batch Log binder.
7.0 / Quality Control
7.1 / The TRP Lab Director or his designee will review form 211-A.
7.2 / The TRP Lab Staff will archive form 211-A.
8.0 / Special Considerations
8.1 / Aseptic techniques will be used at all times. See TRP Document D150.
8.2 / Universal Precautions will be observed at all times.
8.3 / Medium batch numbers will be assigned by using a preparation date code.
8.3.1 / Example: Medium prepared June 3 2002 would be assigned the lot # 060302 (mmddyy).
8.4 / It is critical to ensure that the base RPMI 1640 medium contains Glutamax and HEPES. If not they must beadded separately as described in Attachment 10.1.
8.5 / Until January 2007, 293T Culture medium was prepared using HI&SF FCS.
9.0 / References
9.1 / University of Pennsylvania Biological Safety Manual, 2nd edition, 1995
9.2 / TRP Document D150: Basic Sterile Technique
9.3 / SOP TRP135: Heat Inactivation of Serum
9.4 / SOP TRP210: R10 Culture Medium Preparation
9.5 / SOP TRP340: 293T Cell Maintenance Culture
10.0 / Attachments
10.1 / Attachment 10.1: Formulation: 293T Culture Medium
10.2 / Attachment 10.2: Form 211-A: 293T Culture Medium Batch Log

11.0Annual Review

Laboratory Director Date

Attachment 10.1: Formulation: 293T Culture Medium

A.For RPMI1640 Containing Glutamax and HEPES (e.g.Invitrogen/Gibco Cat. #72400)

Ingredient / Stock Conc. / Final Conc. / 500 mL
Final Volume / 1000 mL
Final Volume
RPMI 1640 / N/A / N/A / 445 mL / 890 mL
FCS (not HI&SF) / 100% / 10% / 50 ml / 100 ml
Pen/Strep / 10,000 IU/ml,
10,000 ug/mL / 100 IU/ml,
100 ug/mL / 5 mL / 10 ml

B.For RPMI1640 Lacking Glutamax and HEPES(e.g.Biowhittaker Cat. #12-167Q)

Ingredient / Stock Conc. / Final Conc. / 500 mL
Final Volume / 1000 mL
Final Volume
RPMI 1640 / N/A / N/A / 427.5 mL / 855 mL
FCS (not HI&SF) / 100% / 10% / 50 ml / 100 ml
Glutamax / 200 mM / 2 mM / 5 ml / 10 ml
HEPES / 1 M / 25 mM / 12.5 ml / 25 ml
Pen/Strep / 10,000 IU/ml,
10,000 ug/mL / 100 IU/ml,
100 ug/mL / 5 mL / 10 ml