PROTOCOL- Fibroblast Cell

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APPENDIX A
  ]+ f: d; i: e( e* O* ?OVERVIEW OF FIBROBLAST MEDIA
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500 ml Bottles (except where indicated)
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+ u0 j  _4 _7 f" g' |6 `2 e7 j7 ICC-3131 FBM?   Fibroblast Basal Medium (no growth factors) ( B1 H& R# ?1 t, Z) n% J
CC-3130 FGM? BulletKit?   Kit which contains a 500 ml bottle of FBM?, (CC-3131) and FGM? SingleQuots? (CC-4134) which contains all of the supplements listed below, conveniently packaged as single-use aliquots (amounts indicate concentration of each SingleQuot?)' i0 R* E4 N- R9 z
1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml
  U, ?% h9 B- k3 O4 M4 }4 z5 mg/ml Insulin (CC-4021) 0.5 ml
: X: n) {  q) }: d7 I0 p50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml6 _, o3 @8 E/ D" q
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CC-3132 FGM?-2 BulletKit?   Kit which contains a 500 ml bottle of FBM?, (CC-3131) and FGM?-2 SingleQuots? (CC-4126) which contains all of the supplements listed below, conveniently packaged as single-use aliquots (amounts indicate concentration of each SingleQuot?)6 \7 S( J' Z& E( n6 Y$ N6 H
1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml
: x/ \0 n( K& j/ u4 A2 A5 mg/ml Insulin (CC-4021) 0.5 ml 0 H; C4 Q5 K, L' A- I
50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml & @9 z- M2 K3 ^' U" l0 G; j
10 ml FBS (Fetal Bovine Serum) (CC-4101)" G# \2 Q+ r  T2 v+ Q5 V

1 _, U' T; P* a. Q5 _' ICC-3134 FGLM™ Custom   FGM? Labeling Medium BulletKit? (500 ml) that consists of the following:
1 h9 z' o. O2 W5 p0 I  CC-3133   FBLM™ Fibroblast Basal Labeling Medium, without the following nutrients: Myo- Inositol, Thymidine, Proline, Isoleucine, Leucine, Methionine, and Cysteine.
& \1 m. A! d2 b/ r) T  CC-4153   FGLM™ SingleQuot? Kit, FGM? labeling SingleQuots? consisting of the following:
5 y# N) c- i( n9 g* M0 V3.513 mg/ml L-Cysteine(CC-4069)2 ml
3 w/ i2 g) p1 W6 j: T6.559 mg/ml L-Isoleucine(CC-4045) 1 ml- C& R6 V9 ~. k
9.01 mg/ml Myo-Inositol(CC-4050) 1 ml/ b; C$ x4 C3 n/ [" {3 d
9.838 mg/ml L-Leucine(CC-4046)2 ml2 O' L* f9 J1 K, J
2.238 mg/ml L-Methionine(CC-4047) 1 ml
, I- Y2 M3 E: Z: [5 J& G* c2.878 mg/ml L-Proline(CC-4048)1 ml: Y1 t* x& b3 e, A3 G7 S
0.036 mg/ml L-Thymidine(CC-4049) 1 ml: `" J- C, d" D1 t* g
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  CC-4134   FGM? SingleQuots? (See CC-3130) & U9 m* @# W1 [2 I' z" t$ Y2 X2 C
CC-3135 FGLM™-2 Custom   FGM?-2 Labeling Medium BulletKit? (500 ml) that consists of the following:
: f7 T0 X6 D/ D4 F8 U  CC-3133   FBLM™ Fibroblast Basal Labeling Medium, without the following nutrients: Myo- Inositol, Thymidine, Proline, Isoleucine, Leucine, Methionine, and Cysteine.
( @! H  z5 M* M7 Y& ~6 \  CC-4153   FGLM™ SingleQuot? Kit, FGM? labeling SingleQuots? consisting of the following:
' w- ?! _5 \$ S1 Y6 ?3.513 mg/ml L-Cysteine(CC-4069)2 ml# R+ |: [/ t6 H: C& {
6.559 mg/ml L-Isoleucine(CC-4045) 1 ml
4 Z. J1 O* r2 d9 N9.01 mg/ml Myo-Inositol(CC-4050) 1 ml) J3 q" ^- g" w) N2 K# r
9.838 mg/ml L-Leucine(CC-4046)2 ml
4 `8 Z0 b: v- d* l+ c3 h  A+ @/ H+ v2 E2.238 mg/ml L-Methionine(CC-4047) 1 ml( ]% {3 ~' ~  K/ E
2.878 mg/ml L-Proline(CC-4048)1 ml$ G5 C, L7 Y; O/ M( @6 a
0.036 mg/ml L-Thymidine(CC-4049) 1 ml
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: m' X* W( M- \0 g# U1 t6 u$ J  CC-4126   FGM?-2 SingleQuots? (See CC-3132) - U* }0 l! ]3 e; p3 C

' G! `; J# }9 ]3 C1 I/ xNOTE: All Clonetics? Media can be custom formulated to meet your research needs. Contact your Technical Specialist for more information.

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BIBLIOGRAPHY
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1) Polymerase Chain Reaction (PCR) technology is covered by U.S. Patents 4,683,195, 4,683,202, and 4,965,188 owned by Hoffman La-Roche, Inc.
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2) Cytokeratin 18 & 19. Call your Clonetics? Technical Specialist for a reference on this procedure.
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3) Wagner, D. D., Olmsted, J.B. and V.J. Marder. (1982) Immunolocalization of Von Willebrand Protein in Weibel-Palade Bodies of Human Endothelial Cells. Journal of Cell Biology., 95:355-360.
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4) Grizzle, W.E., and S.S. Polt. (1988) Guidelines to Avoid Personnel Contamination By Infective Agents in Research Laboratories That Use Human Tissues, J. of Tissue Culture Methods, Vol. 11, No. 4.; O/ X+ O" ^  @5 A
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TECHNICAL SERVICE:' b9 Q1 M6 f6 \  _) u
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Clonetics? Products
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7 {1 k! W/ a* k: j0 C3 |5 gSan Diego, CA 921215 P9 Q% [$ q7 j4 @  J
(800) 852-5663
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' x" C) M" [7 x& I. a9 D; U9 ]/ NINTERNATIONAL TECHNICAL SERVICE:
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BioWhittaker Inc.
7 d& n, l; h6 P) zClonetics Products
, }' V  e1 D* D8 g3 T% X  O* P0 E6 U8830 Biggs Ford Rd.9 ^% H) j  y/ z/ O
Walkersville, MD 21793-01274 L+ @- c* c0 X) q
301-898-7025  K& a. U7 p. q  \. i# W$ q+ v
FAX: 301-845-2924
) g& ]7 s: K8 k# ]E-mail: techsup@biowhittaker.com! ]" W1 e( B& [. I! X

7 b! V( |0 \  Y) GORDERS:' Z7 b9 w, R7 o9 M6 _7 [
BioWhittaker, Inc.
/ e% `* }2 b5 w; ^4 [* g& ~Clonetics? Products6 Y. Y4 a' r8 d& ~. K, G
8830 Biggs Ford Road
/ h6 K0 ?0 M( c3 B; DWalkersville, MD 21793
$ e6 Y  q+ s# K" K(800) 344-6618

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Subculturing
5 |% Q6 M* G9 e% J( D. oExamine your cultures microscopically every day. 6 k1 f2 M" r4 ~
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Subculture the cells when they reach 70-90% confluency. NHDF and NHLF cultures should have many mitotic figures throughout the flask. Cells should be ready to subculture within 24 to 48 hours, however, shipping conditions such as temperature fluctuations may affect the actual time at which the cells are ready for subculture. . O- \  N% u! x9 @7 k2 V4 c
Avoid the loss of your culture due to contact inhibition by subculturing cells at no more than 90% confluency. See pages 15-17 for detailed subculturing instructions.

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Maintenance After Subculturing
* ?& `8 Z  w. s0 {2 W" NAfter 24 hours:
7 w7 V: H+ y/ k0 T3 A. a5 Q1. Examine the cells microscopically. At least 60% of the cells should have attached to the culture flask.
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Some cells will be loosely adherent, but most will have spread out on the culture flask surface. At this stage, most cells will be single or in small colonies.
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1 F, {/ K( t) z' ^/ [& p2. Change the culture medium to remove residual trypsin and non-attached cells. & o0 r6 h2 u* y3 ~7 K
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3. Incubate for an additional 24 hours, and re-examine the culture.
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a.At this stage, the vessel should have several mitotic figures indicating that the cells have resumed active growth.2 Z, |" S; p2 h" ]0 u1 G0 l
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b.If few mitotic figures are observed, contact your Clonetics? Technical Specialist for assistance.
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8 f& K) V# B6 D9 W" W( r4. Change the medium again 48 hours after the day 1 feeding, and every 48 hours thereafter while examining the culture daily.
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& W: d7 `  P  \; J) k& V8 [5. Feed with volumes as outlined in the table on page 13.; m3 x$ u  i$ U+ K5 R. Q

) S  m9 ]$ g/ O6 S: A6. Passage again when the cells are 70-90% confluent. (If seeded at the recommended seeding density, this should take 5-9 days.): v* B! C, @. U; A( l0 M6 f. _" P
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Instructions for Proliferating Cells- U7 S7 C+ I6 a# ^
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Cell Preparation: Proliferating Cells) B; c5 p- g; {
With the proliferating culture of NHDF or NHLF you received, do the following:
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Examine the culture microscopically for any signs of distress during shipment (i.e., detachment, rounding-up or atypical morphology). Check the relative cell density and estimate "% confluency." The culture should be 30-80% confluent upon receipt. Some cellular detachment is normal. Please call Clonetics? Technical Specialist immediately if cells look severely distressed. . v/ m$ @+ D% M/ I2 [
Decontaminate the external surface of the cell culture flask by wiping with 70% ethanol or isopropanol.
$ Y6 M% m2 a6 Y1 r+ EIncubate the sealed flask at 37?C, 5% CO2 for three to four hours to equilibrate temperature.
7 V0 |+ T; T$ m; Q. \7 Z" Z1 |Warm an appropriate amount of growth medium (see table on page 13) to 37?C in a sterile container. Warming the entire bottle can shorten the life of the medium. Never warm growth medium under hot running water or any other uncontrolled temperature source. NEVER MICROWAVE!
8 O' A+ R0 T5 W6 Z. VIn a sterile field, carefully open the cell culture flask, remove the medium and replace it with the warmed, fresh growth medium. Aseptically remove any medium inside the neck or cap area because it can facilitate microbial contamination.
5 b. h6 G* W4 C- s5 y" bLoosen the cap, and return the flask to the 37?C humidified incubator with 5% CO2 for at least 24 hours.

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Assessing Yield and Viability' m$ C- n) v  H/ j+ v1 M
Several factors contribute to low cell count and low cell viability. An example of yield viability assessment is provided in the chart below. To determine the reason for low yield/viability, follow these steps:
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' r" w& i2 H3 I* z2 P: e1. Study the sample chart below. It is a sample of high yield, high viability.
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a. Note the "solid dot" on the far, left side of the square. It indicates high yield, or a cell count of more than 250,000 for NHDF and more than 500,000 for NHLF.$ u, H4 x& C# F9 t! p
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b. Note the "solid dot" on the X axis or bottom line of the square. It indicates high viability, or more than 50% viability.' C( S: q/ |: q+ I0 j; b/ }& p
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c. Extend a line from each dot as shown in the chart. The point where the lines intersect (the bold "X") is located in the High Yield/High Viability quadrant. Thus, the sample is optimal., D' D+ W  y' g) i" x* C$ `( R! x
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2. Now, using the blank diagram below plot your cell yield and cell viability. Follow these steps:# m: o' [# E2 \

4 x/ `* Z5 o3 i! na. Mark a (?) on the Y axis to indicate the total cell count of your culture.6 J; q. C, t  V% Q

& M4 L8 U( H) V3 o8 zb Mark a (?) on the X axis to indicate the calculated percent viability of your culture.
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* K+ j( k9 Y0 u" I( e# j$ k2 }3. If your result falls into any quadrant other than the "High Yield-High Viability" quadrant, refer to Appendix D, Improving Cell Yield and Viability, before proceeding to your next trypsinization.

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Subculture Preparation
( S  \) `; ]; B  b& [4 n0 |# LNOTE: The following instructions are for a 25 cm2 flask. Adjust all volumes accordingly Preparation for other size flasks.. Y! n% S- t' r+ u; r! |( K
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Preparation for subculturing the first flask:
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; d, O# x5 l5 o/ JSubculture the cells when they are 70-90% confluent and contain many mitotic figures throughout the flask. ' L( z: E4 e( D  J5 A% s' y
For each 25 cm2 of cells to be subcultured, allow 3 ml of Clonetics? Trypsin/EDTA (T/E) to thaw and come to room temperature. For NHDF grown in FGM? use cold T/E (4?C) for subculturing.   a6 u6 d# k  q
For each 25 cm2 of cells to be subcultured, allow 5 ml of Clonetics? HEPES Buffered Saline Solution (HEPES-BSS) to come to room temperature.
6 K% T" S' G1 x9 e) R0 tFor each 25 cm2 of cells to be subcultured, allow 3 ml of Trypsin Neutralizing Solution (TNS) to come to room temperature. % G7 S6 \" }0 w6 F. \6 K
Remove growth medium from 4?C storage and allow to start warming to room temperature. 5 |; y  Z/ b+ L$ q; P, L- ?& m
Have flasks available for seeding cells. 2 P' g1 Y+ J: n
Subculturing
% d$ k/ B; E9 J6 H$ Z  Z- OSubculture one flask at a time. All flasks following the first flask will be subcultured following an optimization of this protocol (explained later in this procedure), based on calculated cell count, cell viability, and seeding density. 5 k  @: @. A- F+ }
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In a sterile field:
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Aspirate the medium from one culture vessel. * ^1 G) @$ S8 }! ^* n1 R7 F
For NHLF and NHDF grown in FGM?-2, rinse the cells with 2 - 3 ml of room temperature HEPES-BSS. DON'T forget this step. The medium contains complex proteins that neutralize the trypsin, making it ineffective. For NHDF grown in FGM?, skip steps 2 and 3.
& n( Z* j/ T% }/ o- jAspirate the HEPES-BSS from the flask.
- q; g3 R% c2 T# p9 V( \Cover the cells with 3 ml of Clonetics? T/E solution. Use cold (4?C) T/E solution for NHDF grown in FGM?. 2 c& W6 O" j6 R3 x+ H
Rock the flask to make sure all cells come into contact with the trypsin.
( s! {7 C* y0 N; ^2 G4 BTighten the cap and begin monitoring the flask under the microscope. % o) {" V. F7 e2 `* z
Continue to examine the cell layer microscopically. $ ]. r, P- s2 ]) p8 E
a. Allow the trypsinization to continue until 3 90% of the cells are rounded up.
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" L/ W! n- t6 t: x$ n" ONOTE: Rounded up cells are spherical, have smooth edges and are refractile or shiny. If the cells still have protruding nubs which are still attached to the flask, they need more time to trypsinize. This entire process takes about 1 to 2 minutes, under optimal conditions.
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' T- M* \  t" Q) T, R) i2 h  Tb. At this point, rap the flask against the palm of your hand to release the majority of cells from the culture surface. If only a few cells detach, you may not have let them trypsinize long enough. Wait 30 seconds and rap again. If cells still do not detach, wait and rap every 30 seconds thereafter.
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NOTE: Don't try to get all cells to detach by rapping them severely. This action may damage the cells.; E# e5 ?0 v7 Y" r
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After cells are released, neutralize the trypsin in the flask with 3 ml of room temperature TNS.
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If the majority of cells do not detach within four minutes, the trypsin is either not warm enough or not active enough to release the cells. Harvest the culture vessel as described above, and either re-trypsinize with fresh, warm Clonetics? Trypsin/EDTA Solution (or) rinse with Clonetics? Trypsin Neutralizing Solution and then add fresh, warm growth medium to the culture vessel and return to an incubator until fresh trypsinization reagents are available.
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Quickly transfer the detached cells to a sterile 15 ml centrifuge tube. , G& l' b# E: F) I: v* t6 Q
Rinse the flask with a final 2 ml of HEPES-BSS to collect residual cells, and add this rinse to the centrifuge tube. + m; t2 d& T/ U/ e; a- }# s
Examine the harvested flask under the microscope to make sure the harvest was successful by looking at the number of cells left behind. This should be less than 5%.
8 u% s. P  E8 }* w$ JCentrifuge the harvested cells at 220 x g for 5 minutes to pellet the cells.
# r+ N# h: e( e+ Z3 \; l$ ma. Aspirate most of the supernatant, except for 100-200 ml.
$ B! u8 [. q, vb. Flick the cryovial with your finger to loosen the pellet.: N  i8 Q7 b7 f3 q
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Dilute the cells in 4-5 ml of growth medium and note the total volume of the diluted cell suspension.
0 O- A/ |) d; E$ `* M, RDuring these procedures keep the cells in ice until they are plated." C) P! U" t/ Q

+ ], q4 T2 _: c$ R( ]+ K% LTo obtain the best results from your cells, you will assess cell yield and viability with Trypan Blue. Trypan Blue is a dye used to highlight dead cells. Dead cells take up the dye and appear blue, instead of refractile and colorless. Evaluate on bright-field microscope. Follow these steps:$ |4 ^0 _" {* m* A
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Count the cells with a hemacytometer or cell counter and calculate the total number of cells. (See Appendix B.) Make a note of your cell yield for later use.
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The cell suspension should contain between 250,000 to 1,000,000 cells/ml for greatest accuracy.
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If necessary, dilute the suspension with HEPES Buffered Saline Solution (HEPES-BSS) to achieve the desired "cells/ml" and re-count the cells.
, X8 g2 x% M) T  NAssess cell viability using Trypan Blue (see Appendix C).   p9 G! Z0 N3 t2 w" Q
Use the following equation to determine the total number of viable cells:
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Total # of Viable Cells = Total cell count x percent viability / 1006 j& J7 }( z$ i
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Example: 1,000,000 cells x 60 / 100 = 600,000 viable cells
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; e# U2 \7 ]6 q! H3 ?Determine the total number of flasks to inoculate by using the following equation. The number of flasks needed depends upon cell yield and seeding density. Larger flasks may be used to save plasticware and time spent on subsequent subcultures. Smaller flasks reduce the risk of losing a substantial part of your culture if contamination occurs.
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NOTE: Recommended seeding density is 3500 cells/cm2 for NHDF and 2500 cells/cm2 for NHLF.8 w/ _9 J$ }' Z8 Q' d+ x% O3 K

9 y; I0 J9 v8 dTotal # of flasks to inoculate = Total # of viable cells / Growth Area of Flask x Recommended Seeding Density
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' f8 D- E9 i+ B% P  E$ wExample:600,000 viable cells / 75 cm2 x 3500 cells/cm2 = 2 T-75 flasks (rounded down to nearest whole number)
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& @0 a8 q5 h7 v' K! yUse the following equation to calculate the volume of cell suspension to seed into your flasks.* M% w/ L& H  j9 @
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Seeding volume = Total volume of diluted cell suspension / # of flasks as determined in step 18
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7 v$ d( K! m  h- W. |& y- K4 q/ W. @Example: 4.3 ml of diluted cell suspension / 2 T-75 flasks = 2.15 ml per T-75 flask; [0 Z% F8 C4 m# ]1 s2 {4 m/ t' e

& D. `0 v7 M3 cPrepare flasks by labeling each flask with the passage number, strain number, cell type, and date.
* Z$ ]% d; n" i+ M: m7 RCarefully open the medium bottle and transfer growth medium to new culture vessels by adding 1 ml growth medium for every 5 cm2 surface area of the flask (1ml/5cm2). 6 F  L' {, @+ E3 T2 z. f
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Example:15 ml growth medium for a 75 cm2 flask.5 x7 Q# h; N/ }6 V

* z4 J, J! v* k3 i7 q, ^After mixing the diluted cells with a 5 ml pipet to ensure a uniform suspension, dispense the volume of suspension calculated above into the prepared subculture flasks. 1 f0 |  @7 T5 S$ J, Q/ J
After dispensing the cells, gently rock flask to promote even distribution.
) E* O6 M& u7 m2 ?2 }& J8 @5 kIf not using vented caps, loosen caps of flasks. Place the new culture vessels into a 37?C humidified incubator with 5% CO2..

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Overview of Subculture Preparation
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Thawing % X' u2 i) D; W, r, Z; F# P
NOTE: If more than one cryovial is to be thawed, thaw one cryovial at a time and keep other cryovials in liquid nitrogen until ready for use.8 i: v; t4 G( P9 t( M: k
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After the flasks have equilibrated for 30 minutes:
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0 Q5 Y2 C* F- B0 D* ePrior to thawing, locate a micropipetter.
: s2 e  l( Q) I4 p/ a/ a2 TRemove the cryovial of cells from storage. Wipe the cryovial with ethanol or isopropanol before opening. In a sterile field, briefly twist the cap a quarter turn to relieve the internal pressure, then retighten. Do not open the cryovial completely. ! A, w! N2 i+ W" W6 \
Holding the cryovial, dip the bottom 3/4 of the cryovial in a 37?C water bath, and swirl gently for 1-2 minutes until contents are thawed. Watch your cryovial closely; when the last sliver of ice melts, remove it. DON'T submerge it completely. Thawing the cells for longer than 3 minutes results in less than optimal results. ! K6 Q* U. \0 t
Remove the cryovial immediately, wipe it dry, and transfer to a sterile field where the equilibrated flasks should be waiting, ready to seed. Rinse the cryovial with 70% alcohol, then wipe to remove excess. & T* y6 r: I- J$ Q
Note the color of the thawed cryovial. Ideally, the color of the thawed cryovial should be pink. If the color is not pink, still seed the cells, note the color and mention this fact to your Technical Specialist if seeding is not successful. , q4 T+ B2 l7 T  m# V' N
Seeding
# \0 q  J" v, |. iAfter cells are thawed:
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/ b. W3 j! \& KNOTE: Do not dispense the entire contents of the cryovial into one T-25 flask!!
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Remove the cap, being careful not to touch the interior threads with your fingers.
" E8 G* }' B9 E/ y4 ^Using a micro-pipette with a 1000 ml tip set to 800 ml, put the tip into the cryovial and resuspend the cells, with a gentle, slow and steady up and down pipetting motion no more than five times. DO NOT resuspend quickly, and keep the tip near the bottom to avoid making bubbles.
/ z2 c0 W1 L! W) oDispense an equal amount of cells into the flasks. If five T-25 flasks were prepared, set micropipetter to 200 ml and dispense.
) r% D% Q) ~% c: b6 T% D; V- C' q9 TReplace the cap or cover, and gently rock the vessels to evenly distribute the cells. Loosen caps if necessary to permit gas exchange (see "Set Up," step number 4, pg. 11).
$ M7 @' N& a. M: t" A/ X( X0 @Return the culture vessels to 37?C, 5% CO2 incubator. Lay them flat on the shelf, providing the largest surface for cells to attach. The cells will anchor to the bottom surface of the flask.
2 R6 ^8 V- R) }2 H4 V% t0 Q9 K2 sMaintenance After Seeding( |+ {4 L& p* M3 Y3 r+ ?& t
Normal Human Fibroblasts are not tolerant of rapid temperature fluctuations or nutrient-deficient medium. Feeding them with fresh growth medium that has been warmed will avert potential problems. (Remember to warm only the amount needed.) Check and feed the cells on the schedule below, even on weekends and holidays.; e0 R$ G! `' `( j# L' R
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1. Change the growth medium the day after seeding (to remove residual DMSO and unattached cells), then every other day thereafter while examining them daily. $ _* ]5 r3 y1 R

" I) X: s6 r- U! e& C6 iNOTE: A change of medium requires removal of the medium by aspirating with a sterile pipette on the opposite side of the flask from where the cells are attached. Then warm, fresh medium is added down that same side.; G3 @# x4 k& u. x( E
. u3 V. j2 e- d
2. Successfully recovered cultures will exhibit the following:9 x; y  K1 l" q+ P) u
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a. Cells with clear non-granular cytoplasm.
: M, Z; v8 ]6 g1 q& f/ c. V
/ q8 C/ z2 K. Xb. Numerous mitotic figures after day 2.- h7 b% u7 j- q0 V: c3 p9 t
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3. Feed the cells a larger volume of medium as they become more confluent. Use this table as a guideline:
* I+ N% x' U/ A+ i  x, t
$ o6 u5 I& V. W. s- iIF CELLS ARE: THEN FEED THEM:
$ v! U# I! A+ C  I% _Under 25% confluent... 1 ml per 5 cm2 $ Q/ }  o/ W- d& o3 ~5 b
From 25-45% confluent... 1.5 ml per 5 cm2 0 P& f4 A5 r7 h: M
Exceeding 45% confluence... 2 ml per 5 cm2
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* }! P6 Z1 D8 G' d0 {8 ~4. Continue feeding the cells until 70 - 90% confluence. If the cells are allowed to become over-confluent they will suffer contact inhibition and will pop off the flask and/or be difficult to trypsinize.

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Instruction for Cryopreserved Cells6 f" O1 f8 A: n% H: E

  |, A9 B8 O* l  WBefore You Begin
/ I' L  n0 H) M7 n$ ePerform the following steps before you begin medium or cell preparation:6 Z5 k1 _, ]% V" e* ]
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Step Explanation
) z7 t, \5 M" y& t% \$ @8 c. K+ QPrepare a sterile field. A sterile field consists of a Class II biological safety cabinet with a front access opening and filtered laminar airflow, or other such equivalent device. / a8 V; J/ n, M5 [: F5 M3 L
Determine the amount of medium required. Review the Growth Area of Common Plasticware Chart (Appendix E) to determine the amount of medium to be used.
. Q* B& E: E; p  W( p) NCollect sterile instruments and vessels. Sterile disposable serological pipettes : z, n/ J1 m! d1 O, m3 n1 _
Micropipetters and sterile pipette tips , k( v, I4 I- Q" }* K; a1 m0 {0 {
Adjustable multichannel pipetter or repeating pipetter*
1 z, e7 v0 I; q8 mSterile reservoirs for use with multichannel pipetter* ! i. Q8 J8 Q0 q  _
Sterile 15 ml centrifuge tubes 4 k! B1 X( R9 E* K, M$ G  Z
Cell culture flasks " t$ E. t. ~/ k" |- N( \* j
Multi-well, flat-bottom tissue culture plates* & g# w: p! c5 f9 U; g: m5 e! K
Hemacytometer or cell counter ( J# v' ^5 M7 Q! F# z' ^
- t: F+ z+ T9 H, T: W
Collect other supplies. 70% alcohol (ethanol or isopropanol) ' Q3 A0 W* p6 [" e1 G$ L+ b" n
Growth medium (cell-type specific) 7 F! ^8 R* X9 z! a5 k
Protective gloves and garments
- l9 V' q1 I+ j' u5 nTrypan Blue 8 m* V2 c  }( C4 T

5 P, v3 `. `$ d# l2 TPlan and prepare for initial set up.  Base your set up on the number of cells indicated on the accompanying Certificate of Analysis. (See Appendices B and C.) + }2 Z# Y8 e9 U
Check the calibration on humidified incubator. Incubator should be a 5% CO2/95% air, humidified incubator, set to 37? C.
( _# A9 c. [, K7 l
/ u2 T/ J. P' B# r* May not be necessary for all end-user assays.5 ]% j6 L1 z( D' Z' j) g  E

6 E, A. ]' h) ?# q/ [3 ~Medium Preparation
& j5 [, b; y$ [( N9 h( K3 TPerform the steps below in a sterile field. "Sterile field" is defined above.1 |, |4 i5 g2 b1 W6 ~
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For the FGM? and FGM?-2 BulletKits?, do the following:2 G" ?: [3 p8 f/ e% m* o

6 }$ f1 K: _% C  w. G- HDecontaminate the external surfaces of the SingleQuot? cryovials and the basal medium bottle with ethanol or isopropanol.
, h% G. f6 K, x$ B7 L4 U$ {Aseptically open each cryovial and add the entire amount to the basal medium with a pipette. 0 I  \' s6 L1 _5 s4 _: d' W
Rinse each cryovial with the medium. It may not be possible to recover the entire volume listed for each cryovial. Small losses, even up to 10%, should not affect the cell growth characteristics of the supplemented medium. 7 [5 U) w2 |2 |6 y* X
Transfer the label provided with each kit to the basal medium bottle being supplemented. Use it to record the date and amount of each SingleQuot? added. (We recommend that you place the completed label over the basal medium label to avoid confusion or possible double supplementation.) 8 e0 s+ O! J4 g( ~' M- L  j" [# x. g
Record the new expiration date on the label based on the shelf life (see table on page 6). This supplemented medium will now be referred to as either FGM? or FGM?-2. . u8 [1 p5 G4 {, ^8 m/ \- ]% R
NOTE: If there is concern that sterility was compromised during the supplementation process, the entire newly prepared growth medium may be refiltered to assure sterility. If you refilter, use a sterile 0.2 mm, low protein binding filter. Routine refiltration is not recommended.( |; c+ `2 a# ^$ \) d

( b8 Q+ G3 K: V5 lSet Up
1 U: X- Z3 ~  V/ [0 eTo set up vessels for NHDF and NHLF coming out of cryopreservation, do the following:" P$ |8 @* W% w  L- c+ g! A

4 L( G$ ^5 m1 o& p1. Calculate the number of vessels to be set up. Refer to your Certificate of Analysis for the exact number of cells in your cryovial. Refer to Appendix E, Growth Area of Common Plasticware, for help in adjusting this calculation. 0 [2 |7 U; q2 ?3 x

8 s! h% _7 @9 h9 U& k8 s# ]& c' j5 TNOTE: Flasks and multiwell plates are most effective to subculture these cells.
" }# v4 Z( Q6 S" J3 h8 P) w, }) ]  V% f3 d  K' X, N* Y
Use the following calculations to determine the number of vessels to be set up for the recommended seeding density of 3500 cells/cm2 for NHDF and 2500 cells/cm2 for NHLF.5 g+ }! q3 A  n$ V, m  k0 J/ T

3 W0 ~7 R! I: _1 |0 `2 k; P; g2 E4 T' dNo. of cells available / 3500 cells /cm2 = max. surface area that can be plated
! Z( a  f9 W% H! k. ^+ j7 K  |& U9 V( o
% q% z+ G" q: }Max. surface area that can be plated / Effective growth area of flask = max. no. of flasks that can be set up
  _. z( i+ t! \+ N3 x1 f/ Q2 P) {5 t& B0 T8 J6 w7 t
Example: A cryovial with 520,000 cells, }, J9 K3 ^8 e* ^4 r* ]
520,000 / 3500 = 148 cm24 R7 C0 y1 F6 n; ?9 y* f% v
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If you use a T-25 with an effective growth area of 25 cm2
7 W! e! r; `3 b4 y* L4 U" N' M; }# Q3 U+ Z0 I+ ?/ |
148 cm2 / 25 cm2 = 5 flasks (rounded down to nearest whole number of flasks)# p3 W  V+ a. d) U
% A8 A# Z  m, n6 B- ?# L8 E2 f4 h7 b
A typical cryovial can be plated into at least five T-25 flasks. The advantage of setting up five T-25 flasks from the initial cryovial, as opposed to larger flasks, is that it reduces the risk of losing large numbers of cells. That is, if you experience difficulty trypsinizing the first T-25 flask, there are more T-25 flasks to use.2 U0 ?7 E4 M2 j! v" J: m1 H

: R# Q' E4 D/ g/ o5 i% O2. Label each flask with the passage number, cell type, strain number, and date." m5 j" _0 ]" ^: H5 ]# G. G

  |0 r1 ^$ y6 e6 PExample: For first passage out of cryopreservation for lung fibroblasts with strain number 5099, the label might appear as follows:0 b# V) S1 K3 v$ ]- z" |# K! f  _
: |0 x2 T2 D4 n
3? NHLF 5099; 12/30/963 m% V) P- y3 i2 a: \
) o) \2 F6 O  s! f6 m) x
3. In a sterile field, carefully open the supplemented bottle of growth medium, and aseptically transfer the medium to new culture vessels by adding 1 ml growth medium for every 5 cm2 surface area of the flask.
. @5 c. L& L6 N, K3 M# a1 \
& D9 c" |$ Z- @* sExample: 5 ml growth medium for a 25 cm2 flask or 60 mm plate.
" P( _: P- w# A& S! X8 P) C9 X$ F9 t4 J, n
4. Place caps on vessels loosely if vented caps are not being used (i.e., twist caps until tight, then loosen about * turn). Allow the culture vessels to warm and equilibrate in a 37?C, 5% CO2, humidified incubator for at least 30 minutes.

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5 ?& i* H/ b4 Y: q( }$ `9 c# @; J4 P
Safety Precautions  h4 R' u' U1 O) _
BioWhittaker stresses the importance of the following precautions:, W# d/ ]; }/ G0 F  s
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Safety Precautions + K) e. @; {  R3 g! y! ~1 S
As a precaution against contamination, follow all procedures for handling products of human origin outlined in "Guidelines to Avoid Personnel Contamination By Infective Agents in Research Laboratories That Use Human Tissues," from the J. of Tissue Culture Methods.4 (See Bibliography, page 21.) 5 l* r$ W0 P' q* F7 V1 [- {6 m# P1 a
Always wear gloves and safety glasses when working with all materials. Exercise caution when working with cryopreserved cells; rapid temperature changes may cause splattering of liquid nitrogen.
' G  K# b7 z- ]Wash hands thoroughly after performing all procedures. # o! |- v/ v* C. z
Never mouth pipet.
# W) H& I' I$ _# I1 h. \Do not smoke, eat or drink in areas where reagents or cells are handled.
, |! H3 h. o/ }4 PProducts of human origin are potentially biohazardous. Although each cell strain tests negative for HIV-1, hepatitis B and Hepatitis C, proper precautions must be taken to avoid inadvertent exposure. $ r; a# V1 @0 B7 S- Y8 w) C, R/ ~

- ]# }& q) a; _) \$ B& B% W* e+ AThe flow chart on the following page illustrates the culture process. It is followed by the step-by-step instructions...6 B; ?% S# S/ a( i4 R