PROTOCOL- Fibroblast Cell

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THESE INSTRUCTIONS APPLY TO ORDERS CONTAINING THE FOLLOWING CELL PRODUCTS
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Cryopreserved Cells (Single donor)' x3 t+ ^0 z, p" ^$ ?! U( E1 r! D

9 Q$ h/ {0 |1 s  T$ P0 p8 oCC-2511 NHDF -Ad   3 500,000 cells/cryovial
9 J+ m2 j! D' p! s% M* E0 ICC-2509 NHDF -Neo   3 500,000 cells/cryovial
6 x! O/ L/ p3 R1 H1 ]& ]2 jCC-2512 NHLF   3 500,000 cells/cryovial
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Proliferating Cells
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  n$ |; O5 l/ tCC-2611 NHDF -Ad T-25 flask    Yield: 500,000 cells
8 @  ?9 W) |) G9 a$ N" CCC-0252 NHDF -Ad T-75 flask    Yield: 1,500,000 cells $ b, b3 K- K- v' o. ?
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CC-2609 NHDF -Neo T-25 flask    Yield: 500,000 cells ) B5 X' w2 Y7 m
CC-0210 NHDF -Neo T-75 flask    Yield: 1,500,000 cells 0 W$ ~# m" Q& C( y6 D! ~7 J
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CC-2612 NHLF T-25 Flask    Yield: 1,000,000 cells 7 j! y9 q" q% A3 @
CC-0282 NHLF T-75 Flask    Yield: 3,000,000 cells

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Proliferating Cells in Preseeded? 96-well Plates$ D' D+ g- M# l* d# c
CC-0160 NHDF -Ad   96 wells / z/ S4 C+ I& b" W$ b! c; v+ z5 k
CC-0116 NHDF -Neo   96 wells  1 R  W$ r: f4 I2 s) `' w. Z, R
CC-0164 NHLF   96 wells
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  k' H& J4 x/ A% L/ ACheck all containers for leakage or breakage. Fibroblasts are available in 6, 12, 24 and 48 well plates, T-150 and T-225 flasks. Please call your Technical Specialist for details and also prices. 5 O" g/ S! W, S# w$ @) Q
For cryopreserved cells - If there is dry ice left in the package, place cryopreserved cell cryovials immediately into liquid nitrogen. If no dry ice is left in the package, thaw and use them immediately.
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For proliferating cells - Swab down the flask of proliferating cells with 70% ethanol or isopropanol, then place the flask in 37?C, 5% CO2, humidified incubator and allow to equilibrate for three to four hours. After cells have equilibrated, remove shipping medium from the flask following instructions on page 20.
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3 C- M1 t  ]/ o+ E$ q3 GStore cell culture medium in a 4?C refrigerator.$ E! e6 {% Q' ?) l$ P% b) X

' w1 a& O- o0 v1 Q, _& A/ C% U0 s3 a) hIf you plan to proceed within 3 days, store all growth supplements, HEPES Buffered Saline Solution (HEPES- BSS) and Trypsin Neutralizing Solution at 4?C. Trypsin/EDTA Solution has a limited shelf life or activation at 4?C. If, upon arrival, Trypsin/EDTA is thawed, immediately aliquot and refreeze at -20?C. If frozen, store at - 20?C. If you do not plan to set up the cell culture within 3 days, store all growth supplements and subculture reagents in a -20?C freezer.
" a1 B8 n  _" Y* V8 `9 D9 `, a$ KPlease read and follow these instructions carefully and completely. BioWhittaker is not responsible for product loss due to improper receipt and handling of its products by customers. Replacement product will be sent at the customer's expense.
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*AA-1005*
8 B, I: A( D* ?7 XAA-1005-1 Rev. 04/98
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Fibroblast Cell System
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1. Normal Human Dermal Fibroblasts and Normal Human Lung Fibroblasts from single donors, as either:
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7 _/ Q8 Z) R2 |) A9 m, ?3 @7 }Product Name Normal Human Cell Type Cryopreserved And Proliferating Cells Product Numbers Recommended Media
5 A( @  j+ H5 u4 z; @- z9 A/ xNHDF
2 a# X. }1 q# p0 K2 W) ^+ i0 G+ B3 e( |Adult9 t1 \( \4 Z/ A/ e8 `1 ]
Adult Dermal Fibroblasts  Cryopreserved CC-25114 w3 g9 [+ _" A# S, O
Proliferating T-25 Flask CC-2611
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Proliferating T-75 Flask CC-0252
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Proliferating 96-well PlatesCC-0160
! L0 ?( I/ H( Q, X FGM?-2 BulletKit?
3 H  K+ j5 ~" \! @CC-3132
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* ]5 |4 B# N, y0 x/ l: wNHDF
0 P5 J3 k4 H' n- l3 r) S8 j3 ~9 cNeonatal
8 V3 D2 }  Y: L4 c6 J* @ Neonatal Dermal Fibroblasts  Cryopreserved CC-25096 r& e; @- T  ]* w
Proliferating T-25 Flask CC-2609. B* t4 \8 y) J: a& E6 q. j( E$ p
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Proliferating T-75 Flask CC-0210! Q' d5 V' ]# Z5 i

, w6 F! l+ y9 d* g! s* ]  t$ ZProliferating 96-well PlatesCC-0116; H( y8 v% n$ ]+ Z. u* b. L9 Y
FGM-2? BulletKit? # e" M/ S6 h0 F! y) ?
CC-3132/ H' O3 A) w% m4 Q& K5 J0 n" v' [

5 @1 B& a& n. A# K$ ]NHLF
( `0 F* h- l# ]0 w6 IAdult
# k  o( g* D* s3 ~! A Lung Fibroblasts  Cryopreserved CC-2512
, i" Q  D% H; C/ ^' k; }3 EProliferating T-25 Flask CC-2612
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% _2 S7 z1 E+ X+ |: pProliferating T-75 Flask CC-02828 e8 V  R4 n, Y
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Proliferating 96-well Plates CC-0164, e3 k7 s5 P3 p; c7 _* |) ]6 N
FGM?-2 BulletKit? ! m6 q! F1 q  j. W+ K+ ^. h  o
CC-3132( a) ^4 X. N; ^8 ~0 f

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) h; p/ G7 J8 |% L* kThe proliferating cultures are shipped in flasks or plates filled with medium. The cells should be between 30 and 100% confluent upon arrival. A Certificate of Analysis is provided with each cell strain and indicates QC performance results and donor information.% s) d2 B5 {& Y8 Z' u; {

* ?' g- o9 h3 b9 ?' b5 ^The cryopreserved cultures are shipped in a screw cap cryovial containing approximately 500,000 cells. A Certificate of Analysis is provided with each cell strain and indicates date of cryopreservation, QC performance results, donor information and the number of cells contained in the cryovial.

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2. Fibroblast Growth Medium (FGM?), as either:
/ l) J. o$ ]" @& b; D6 N! UFibroblast Growth Medium BulletKit? (FGM? BulletKit?) (CC-3130), which contains a 500 ml bottle of Fibroblast Basal Medium (FBM?) and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots? (amounts indicate concentration of each SingleQuot?), l2 C8 G& o0 z5 h  d
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1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml/ l4 _' ~7 F* }; X
5 mg/ml Insulin (CC-4021) 0.5 ml/ [. i2 [0 l$ {9 W# _) W
50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml
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Fibroblast Growth Medium-2 BulletKit? (FGM?-2 BulletKit? ) (CC-3132), which contains a 500 ml bottle of Fibroblast Basal Medium (FBM?) and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots? (amounts indicate concentration of each SingleQuot?)
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1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml' Q( q5 t  h4 N6 u
5 mg/ml Insulin (CC-4021) 0.5 ml
* D3 q6 \8 ^8 D% v50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml8 o: _% @9 P5 U" T
10 ml FBS (Fetal Bovine Serum) (CC-4101)
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, k4 K! S$ A+ t$ a7 w3. ReagentPackTM (CC-5034) contains one 100 ml bottle of each of the following subculture reagents:
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, ?% v7 Z/ l1 k9 F, c0 sHEPES Buffered Saline Solution (HEPES-BSS) (CC-5022)1 x 100 ml bottle' A% e4 Z  P1 |. u
Trypsin/EDTA Solution (T/E) (CC-5012) 1 x 100 ml bottle
7 k' Z+ ~# K) [: d! FTrypsin Neutralizing Solution (TNS) (CC-5002) 1 x 100 ml bottle6 R7 K, I3 P6 n" T
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NOTE: If you use a different Clonetics? medium, see Appendix A.) ?# Y) Z5 P% v! Q+ @

2 U3 ?1 c1 ]. R. w! ^, S% ^General Information5 e- Z5 i0 k, d2 }
Product Applications1 |( Y' m( M7 j7 O: @8 Z$ z, z
Clonetics? Normal Human NHDF and NHLF are:5 g  n; A, c% e8 U, F; P/ i

% i% P6 P' e( W! K' dFOR RESEARCH USE ONLY. $ T  g2 `# C/ l, t" _
NOT approved for human or veterinary use, for application to humans or animals, or for in vitro diagnostic procedures.
1 W' J$ L; H" S% EMaterials Not Provided5 p" l# u1 `6 F: f
Fibroblast Cell Systems do not contain plasticware, glassware or other laboratory equipment used in a cell culture laboratory. Individual components are available separately.% H4 U# L0 @9 j7 f2 R
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Product Warranty
" A% t  s7 Y" [) s+ c" \! nCULTURES HAVE A FINITE LIFESPAN IN VITRO. BioWhittaker warrants its Clonetics? cells in the following manner only if Clonetics? media and reagents are used.# J8 i& p; h; n1 F) ]) t7 o

$ s4 }8 U+ R, a2 _  I2 Y2 pNHDF and NHLF cryopreserved cultures are assured for experimental use for fifteen population doublings. % D; M; n; o+ a5 D) I% O
NHDF and NHLF proliferating cultures are assured for experimental use for ten population doublings.
+ L. ]2 E7 b- L; W0 o! v; q; Z8 ^Additional population doublings and subcultures are possible, but growth rate, biological responsiveness and function deteriorate with subsequent passage. 2 D8 y! N% S, y8 g* [- l( @# I: ]
NHDF and NHLF can become irreversibly contact-inhibited if maintained at confluence for more than two days. To avoid the loss of your cells and forfeiture of your warranty, we recommend that you subculture cells before they reach 90% confluence.
4 T. M, b- p. D2 l2 \9 ^Cell Isolation # f  s! A3 @# }* H; u. e
Fibroblast cultures are established at BioWhittaker's cell culture facility from normal human tissue.% q. ^7 y" T; G: P2 {( |

1 M+ w& G! @" B8 g- B7 }NHDF are isolated according to proprietary procedures, incubated until they approach 90% confluence, harvested and cryopreserved as frozen primaries.   E! G# e" m! _3 Q
NHLF are isolated according to proprietary procedures, incubated until they reach 90% confluence, subcultured once and cryopreserved as frozen secondary cultures.
+ n9 o8 Q* n. P& R! b$ R# T  n% QNHDF and NHLF are cryopreserved in FGM? (or) FGM?-2 supplemented with 10% v/v fetal bovine serum and 10% v/v dimethylsulfoxide as a cryopreservation solution to improve cell viability and seeding efficiency upon thawing.

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2. Fibroblast Growth Medium (FGM?), as either:
/ X& _: v0 v1 i' o& l3 x/ S  a  dFibroblast Growth Medium BulletKit? (FGM? BulletKit?) (CC-3130), which contains a 500 ml bottle of Fibroblast Basal Medium (FBM?) and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots? (amounts indicate concentration of each SingleQuot?)
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; x. Z) d* P9 z0 E# x7 R' E1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml
5 Y9 n/ d4 m# C1 {. M) Z' |5 mg/ml Insulin (CC-4021) 0.5 ml
) P7 x* F" i, l% x8 s1 @" p# }/ W50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml+ S5 ]( V( }8 n! F1 p% i" D. I3 \
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(or)6 O' y' Q# _! ~$ u2 @7 H9 ^) e0 V+ ~* g7 L
Fibroblast Growth Medium-2 BulletKit? (FGM?-2 BulletKit? ) (CC-3132), which contains a 500 ml bottle of Fibroblast Basal Medium (FBM?) and all the supplements listed below, conveniently packaged as single-use aliquots called SingleQuots? (amounts indicate concentration of each SingleQuot?)
! g( D7 D6 z9 C" L9 R8 P+ b# m5 @' R' F) R3 ?- V$ v
1 mg/ml hFGF (human recombinant Fibroblast Growth Factor) (CC-4065) 0.5 ml
. ]/ |% h! `) z1 [/ z5 mg/ml Insulin (CC-4021) 0.5 ml
' \& J& @& g5 M: O" R( |50 mg/ml Gentamicin, 50 mg/ml Amphotericin-B (CC-4081) 0.5 ml1 W( v4 p( M; |0 V
10 ml FBS (Fetal Bovine Serum) (CC-4101)
. j' N" _+ m5 D! e: l) x
/ [9 t5 P  A  n$ G5 U) @* z/ U2 V3. ReagentPackTM (CC-5034) contains one 100 ml bottle of each of the following subculture reagents:
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HEPES Buffered Saline Solution (HEPES-BSS) (CC-5022)1 x 100 ml bottle2 b& S0 ~3 d2 I! h& x
Trypsin/EDTA Solution (T/E) (CC-5012) 1 x 100 ml bottle. M; S$ u1 N- r2 W' Y
Trypsin Neutralizing Solution (TNS) (CC-5002) 1 x 100 ml bottle
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NOTE: If you use a different Clonetics? medium, see Appendix A.
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, Z3 S' p% C0 E4 P# J) S, h% u- U4 VGeneral Information- o+ b) U, G4 @/ }' s6 p& b6 {
Product Applications
8 z$ l8 \5 q' j/ y+ h' kClonetics? Normal Human NHDF and NHLF are:* w# p% V* g& R+ Q% @% U$ m

6 M* g  R2 w, \  O4 t; U! U5 HFOR RESEARCH USE ONLY. 6 A) o4 v/ W# D, v, f$ i: Q
NOT approved for human or veterinary use, for application to humans or animals, or for in vitro diagnostic procedures. # L; B3 G0 p, ~7 o/ [" M$ l, \
Materials Not Provided
2 E( j5 b& V6 M" s: ~8 ~: AFibroblast Cell Systems do not contain plasticware, glassware or other laboratory equipment used in a cell culture laboratory. Individual components are available separately.! A( t9 p  k! g1 e6 ^
! w9 ]+ ]5 E0 I
Product Warranty : Y) u; w1 @: D( C' ^# o
CULTURES HAVE A FINITE LIFESPAN IN VITRO. BioWhittaker warrants its Clonetics? cells in the following manner only if Clonetics? media and reagents are used.' T+ a4 x$ D, b" ~

% _: C3 K1 E5 U; N0 x; i9 S+ WNHDF and NHLF cryopreserved cultures are assured for experimental use for fifteen population doublings. 5 |. H, q3 R+ M" o5 T* ^# ~% q
NHDF and NHLF proliferating cultures are assured for experimental use for ten population doublings.
- c: C: X; ~/ g/ O7 _Additional population doublings and subcultures are possible, but growth rate, biological responsiveness and function deteriorate with subsequent passage.
* c9 z1 g& N( L) G  UNHDF and NHLF can become irreversibly contact-inhibited if maintained at confluence for more than two days. To avoid the loss of your cells and forfeiture of your warranty, we recommend that you subculture cells before they reach 90% confluence. ( C% E! E/ l) S
Cell Isolation
/ K4 p, T. Y: l  J3 Y1 j' iFibroblast cultures are established at BioWhittaker's cell culture facility from normal human tissue.
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NHDF are isolated according to proprietary procedures, incubated until they approach 90% confluence, harvested and cryopreserved as frozen primaries. 2 b/ \. A% r! |. j) f* P' s; T; S
NHLF are isolated according to proprietary procedures, incubated until they reach 90% confluence, subcultured once and cryopreserved as frozen secondary cultures.
; L2 t9 Z1 d1 b* z1 J; ~NHDF and NHLF are cryopreserved in FGM? (or) FGM?-2 supplemented with 10% v/v fetal bovine serum and 10% v/v dimethylsulfoxide as a cryopreservation solution to improve cell viability and seeding efficiency upon thawing.
6 E7 \" c" t- p* [Medium Information
; |8 Q3 a7 V  F" Y: ?& qFGM? BulletKit? (CC-3130) and FGM?-2BulletKit? (CC-3132) 2 t. A& ]: |4 ^" |9 a" Q
How Prepared Storage Requirements Shelf Life
3 S9 N. n: w! }All FGM ? BulletKit ? and FGM?- 2 BulletKit ? components have been human cell culture-tested. All solutions are sterile-filtered by passage through a 0.2 mm filter. Basal medium is stored at 2? to 8?C, and growth factors are stored at -20?C until shipment. If thawed upon arrival, growth factors can be stored at 2? to 8?C and added to FBM ? within 72 hours of receipt.* i5 u  x5 o! n1 i8 {- o; B
If thawed and will NOT be used within 72 hours, growth factors must be refrozen. They may be refrozen only once and then stored at -20?C for up to one year.$ C* ~# _' y# ]1 G  r0 r& Z
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Store FBM ? and FBM ?-2 at 2? to 8?C Store fully supplemented FGM ? and FGM ?-2 at 2? to 8?C. " V5 u% d7 A. _

* p  v# x) l  V" E( _( u' m6 N# DAvoid repeated warming and cooling of the medium. If the entire contents are not needed for a single procedure, transfer only the required volume to a sterile secondary container. Do not freeze. 2 M2 d9 Z! O/ |" q( J* h. k+ c; H
FGM ? BulletKit ? and FGM ?-2 BulletKit ? shelf life are limited by the shelf life of the FBM ?, which is 1 year from the date of manufacture. When growth factors are added at any time within this time period, we recommend use within 1 month, but before the basal medium expiration. : [+ @/ B6 E7 ]

3 m- y/ N( n7 e: _2 Z  x4 Z) nQuality Control % N3 z  ]6 a4 S, W0 v
NHDF and NHLF are cultured without antimicrobial agents and assayed to ensure the absence of microbial contamination after cryopreservation.
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% C% H- P- ?: v8 ^0 r7 oAll cell strains test negative by PCR(1) for HIV-1, hepatitis-B and hepatitis-C. 0 g& Q2 D% h& Z1 z- T% _' f
After recovery from liquid nitrogen, cells are tested for viability, growth rate, morphology, seeding efficiency, proliferative capacity, mycoplasma, yeast, fungus and bacteria. Each culture meets Clonetics? product specifications for proliferative capacity, (i.e., 15 cumulative population doublings after thaw). , T2 x+ Z- r) i# p  ~; F6 G; s
Additional Testing: 3 g$ _) e& k' X! U1 u
Cell Type  Von Willebrand Factor  Smooth Muscle Alpha Actin  Cytokeratins 18 & 19 0 Z) v) {; B. p2 x/ k( K
NHLF Negative Negative Negative ( S+ Z- k: z# I6 W" |5 ^- Q# |
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Before shipping, all basal media, growth factors and cell culture reagents are tested for sterility and cell culture performance.
6 D4 z, T. ?7 I* l9 v* c4 @Subculture Reagent Storage9 |% S6 B6 F6 Q) ]
1. Subculture reagents are stored at -20?C until shipped from BioWhittaker's Distribution Centers.! x) j; a4 {4 [
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2. Subculture reagents may thaw during transport. They may be refrozen once.! [- o( D. Z; u) Y7 Z

4 _& F. E, e6 Z% p  I3. Subculture reagents can be stored at -20?C for up to one year after thawing once and refreezing.
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4. To keep Trypsin/EDTA fresh and active after thawing, you should aliquot it into five 20 ml sterile centrifuge tubes and refreeze at -20?C. Trypsin/EDTA may be stored frozen up to one year.
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5. We recommend that HEPES-BSS and the Trypsin Neutralizing Solution, once stored at 4?C, be used within one month.
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Handling Precautions
8 ], j* B. Q/ n: ~" m. F2 G; a; pNormal human cells are fragile, and require special handling:
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& r6 z) s' u8 |Upon receipt, immediately store cryopreserved cells in liquid nitrogen. Properly stored cells remain viable indefinitely.
0 h( q$ K( ?- ]7 ]$ N: UUpon receipt, immediately place proliferating cells in a 37?C, 5% CO2, humidified incubator. 6 _6 ?4 q2 `9 v$ G% H
Do not use the medium or reagents beyond the expiration date. : y. a) e, N8 W* g
Normal human cells are very sensitive to impurities in commercially available Trypsin. Use only Clonetics? Trypsin; every lot of our Trypsin is tested on normal human cell cultures.
! q3 E* @) D! [  fUse only Clonetics? media. Keep media refrigerated at 4?C. When using a medium, take just the amount you need and then return the bottle to the refrigerator.
2 W- n. `0 c: f2 }2 ]Regularly wipe flasks, cryovials, bottles and gloves with 70% isopropyl alcohol or 70% ethyl alcohol. 9 R# L- j- _' {9 D( o' |
Because cells are anchored to one side of a flask, always add all liquids by pipeting them down the opposite side from where the cells are attached.

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- @2 i- w$ F3 B. Q: h0 |8 |Safety Precautions
& o5 D$ A% ?2 ?; gBioWhittaker stresses the importance of the following precautions:
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Safety Precautions
3 C+ }, u4 O, Q2 B% [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.)
3 [5 B3 u, `3 c( D+ OAlways 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. . |9 n5 U( L6 d+ M& e+ f
Wash hands thoroughly after performing all procedures.
+ X# p! M5 K  k; c7 WNever mouth pipet. ; }8 A3 ?5 W+ c4 Q
Do not smoke, eat or drink in areas where reagents or cells are handled. + p* ?# ]: P( M+ J* C
Products 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. . t: d3 j* K. R; y4 X9 V

" T" g# j7 W' T- n" NThe flow chart on the following page illustrates the culture process. It is followed by the step-by-step instructions..., X1 e! j; x" p7 R8 W- s

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Instruction for Cryopreserved Cells
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Before You Begin. }1 j$ S8 s+ I( U
Perform the following steps before you begin medium or cell preparation:7 D2 Y2 X: }1 g, ?

7 Z9 Z! T* z+ y4 ^/ _. FStep Explanation , `) O$ g' o) T2 @1 P8 ^* G
Prepare 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.
% `. s* J" G8 L! dDetermine the amount of medium required. Review the Growth Area of Common Plasticware Chart (Appendix E) to determine the amount of medium to be used. # L! ~$ K  C; E; u; G% T
Collect sterile instruments and vessels. Sterile disposable serological pipettes 4 D& i( z: n" X+ D. q- ^' v
Micropipetters and sterile pipette tips & t: K; S3 C1 V/ q
Adjustable multichannel pipetter or repeating pipetter*
8 }7 {5 K6 ]5 S# lSterile reservoirs for use with multichannel pipetter*
5 j4 W6 p1 S3 a! v- CSterile 15 ml centrifuge tubes
3 s/ i; t( c' c1 }+ d. fCell culture flasks   J. v% u5 F4 W* `/ h2 s5 T7 q
Multi-well, flat-bottom tissue culture plates* ; i7 Z% ~/ R3 W  e7 _( ~8 H' Q
Hemacytometer or cell counter
0 S% h" Q, n- z5 r  C7 ?5 k  B ( p- ~; k, G  f/ N+ s
Collect other supplies. 70% alcohol (ethanol or isopropanol) ; ~- a6 U2 J- H7 s; J8 f
Growth medium (cell-type specific) 4 z! V$ e/ g% n4 U/ r, N. x
Protective gloves and garments
7 j8 |0 i3 K0 m: b: R& P: C3 cTrypan Blue
& A: h3 O4 T( {$ P4 i: Y" W
( F; U/ q/ O5 l" E5 [+ M9 l. h! WPlan 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.) & {: R7 L0 _& s5 A6 z
Check the calibration on humidified incubator. Incubator should be a 5% CO2/95% air, humidified incubator, set to 37? C. % x  c) P/ C6 H
2 Z) R( o+ j1 O4 M) K0 ?
* May not be necessary for all end-user assays.1 r- g' i& R0 f4 P
; m+ A- W5 k7 G0 ], E. r3 e
Medium Preparation
" Q6 E( i4 Y1 X% J* J/ {Perform the steps below in a sterile field. "Sterile field" is defined above.
- _$ J2 B: W2 |; L6 n+ Z, B8 B
/ ~& W3 W" i4 Z/ mFor the FGM? and FGM?-2 BulletKits?, do the following:: U4 A' z- M; O3 g9 l

. C6 j' i$ ^8 S: h4 sDecontaminate the external surfaces of the SingleQuot? cryovials and the basal medium bottle with ethanol or isopropanol. ; L( Y5 u0 v- y
Aseptically open each cryovial and add the entire amount to the basal medium with a pipette. 8 e5 [, m( @' z( \
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. 6 }5 B. ^' ?/ A, D" A; A5 p
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.) ; p; p1 z0 e( t8 k
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. ; d3 `) i8 t6 U% x7 _+ A
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.
2 S3 r' j* r, n5 ?0 K! Z0 R- ?5 w. u. U  o# g& q; b
Set Up . f6 L% p7 ]5 `1 ?
To set up vessels for NHDF and NHLF coming out of cryopreservation, do the following:
! U* O+ V% J/ J1 d8 v$ n( A, b* O
' s' U' b* }8 G6 L2 v7 r1. 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.
  ?8 V& n9 j, G/ A0 k# k: Y
2 A2 h* Y* n) c# ]( i; B) I5 xNOTE: Flasks and multiwell plates are most effective to subculture these cells. 8 q( S+ ?% Z2 ?) f
  s2 V2 D) g: U3 a) o* T1 B6 Z
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.
6 ]1 U0 q+ ]/ d. a- b. c* ^) h6 \4 G. Q- U# L
No. of cells available / 3500 cells /cm2 = max. surface area that can be plated
  e  H1 k! K8 V$ ^: T% }# _" Z- f* B" N: J0 `  @* i
Max. surface area that can be plated / Effective growth area of flask = max. no. of flasks that can be set up
4 G, j! m+ L* l+ M* |: v
% r3 E9 _! j% `& ^- l4 w  {Example: A cryovial with 520,000 cells
; I/ h1 I. w8 o3 x520,000 / 3500 = 148 cm2/ m: a4 Z. a. }! x# s

. u0 m& l8 g, I$ BIf you use a T-25 with an effective growth area of 25 cm28 W! e' U9 Z' h% o

$ f+ W% f1 |& r148 cm2 / 25 cm2 = 5 flasks (rounded down to nearest whole number of flasks)
: I& J. Z. ]; G0 H7 u' |* H3 R) a( N" X1 H& J& C' f; q, m7 _
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.  e1 y2 |* ?; u: |

9 }- P6 G5 `- n% M  T) i, C, d2. Label each flask with the passage number, cell type, strain number, and date.$ h% K) t* u: p. `8 H" Y

' t: N/ C7 i9 ]1 O( gExample: For first passage out of cryopreservation for lung fibroblasts with strain number 5099, the label might appear as follows:
1 k& m2 H; ^3 x6 [
9 r3 h* r+ Y$ t! O3? NHLF 5099; 12/30/96
; f( [. X9 h$ D1 @5 W5 ?" m3 s3 w6 P
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. : ?8 G- `6 B5 d, d* i
9 i, U8 z" N% h  p5 X" o
Example: 5 ml growth medium for a 25 cm2 flask or 60 mm plate.2 I% R5 g7 t3 l

9 M6 ?* \2 @0 {8 }) M4. 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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Thawing * R& |2 T6 E5 z# y8 |& B( Q; l
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.
' L0 j& M5 L5 u9 g. ^7 p- f* S6 D5 f" B# }8 Q
After the flasks have equilibrated for 30 minutes:/ T5 i+ k8 R+ X1 Y4 E3 _! d

- @' ]1 [) T6 M7 ]9 i) A+ mPrior to thawing, locate a micropipetter.
  E# G& Y6 k  P4 \Remove 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.
1 H, o( o* ]) ?9 Z' d# ]1 y. I  ]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.
* x0 n7 d. D; ~$ u3 b& _7 X% ZRemove 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.
6 K- i* r8 S" i: M, BNote 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.   N; L9 x: }7 U  b( m
Seeding ' c4 @. }  P* [6 V
After cells are thawed:
# q; T: Q+ s% y& e) Z
% [' m5 z/ v2 C3 x/ N: INOTE: Do not dispense the entire contents of the cryovial into one T-25 flask!!
: s; l/ X: Q( P9 |. {9 Y' @8 N0 @/ G2 ]  S- z1 N* f# A& R
Remove the cap, being careful not to touch the interior threads with your fingers.
- V* |0 L" b4 B1 X; Z) aUsing 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. : x5 u5 |6 O7 q, {% k6 f: b; L6 }* Q- f
Dispense an equal amount of cells into the flasks. If five T-25 flasks were prepared, set micropipetter to 200 ml and dispense.
1 g! H  W& l, [Replace 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).
: T2 e4 R7 `1 b2 i2 O; Q% R+ ?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.
) D7 H+ z; N- w4 q+ q: PMaintenance After Seeding( W( v& k: T1 |* e8 @
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.$ ^' T$ k/ q; m2 {" W  ~$ Z

, T, {) h( E3 Q; J1. Change the growth medium the day after seeding (to remove residual DMSO and unattached cells), then every other day thereafter while examining them daily.
0 f! u3 l$ L$ @' J' X" U1 o0 A
4 n2 Z+ k% N$ x/ J$ FNOTE: 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.$ o6 ^& @) Z) i) @' W

: L4 _8 c* K, R" ]2. Successfully recovered cultures will exhibit the following:4 o: B: a; b! v2 ^/ W
* `6 L$ J" o# T- F  u0 V/ J3 C
a. Cells with clear non-granular cytoplasm. " P( h- v" y" Y! a
" S4 d7 S9 K: ]6 Y
b. Numerous mitotic figures after day 2.
! B5 N. j. J0 Q
( ^* x1 M/ X7 c, ?5 n3. Feed the cells a larger volume of medium as they become more confluent. Use this table as a guideline:  x4 s1 v: S0 a5 U
* C' X1 y; f& z" J
IF CELLS ARE: THEN FEED THEM:   U# O3 c/ J# _, n7 r
Under 25% confluent... 1 ml per 5 cm2
- R4 I  A* G, M% l6 g! W. R- e7 yFrom 25-45% confluent... 1.5 ml per 5 cm2
( }! T0 f8 U' H6 R1 u5 tExceeding 45% confluence... 2 ml per 5 cm2 % T9 t1 h3 F7 [$ a
: h% w6 b) }. `( `
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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Overview of Subculture Preparation7 O1 v6 P8 ~+ g$ B

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Subculture Preparation
/ ]4 T- o" H) C: O$ SNOTE: The following instructions are for a 25 cm2 flask. Adjust all volumes accordingly Preparation for other size flasks.
0 W5 G7 `& o$ M( n! n# D  F5 L8 r& j! Z8 l) z$ X
Preparation for subculturing the first flask:
' B) O# `- ]+ F! f2 B6 Z% y. a. R* Q/ u. m
Subculture the cells when they are 70-90% confluent and contain many mitotic figures throughout the flask.
& C; c6 v. l6 L' _% uFor 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.
4 S: i" e# u( s/ SFor each 25 cm2 of cells to be subcultured, allow 5 ml of Clonetics? HEPES Buffered Saline Solution (HEPES-BSS) to come to room temperature.
+ v" s& X& }; ^4 p9 T- S: A% [For each 25 cm2 of cells to be subcultured, allow 3 ml of Trypsin Neutralizing Solution (TNS) to come to room temperature.
1 l$ f* \5 ^1 k. s8 iRemove growth medium from 4?C storage and allow to start warming to room temperature.
# ?$ A4 m. D8 ]# }Have flasks available for seeding cells. $ v& V+ P9 n" u) m
Subculturing
  Y1 U( O! x, U2 b1 X1 O6 T7 d# `. QSubculture 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.
' v5 _% S& n: T
6 S0 Z6 d" O# v- l! J; b% o9 CIn a sterile field:7 w4 C3 T2 E$ e1 X  F* C

' r- G( t8 Z) W# t" ], mAspirate the medium from one culture vessel.
6 @* x* j: n$ `3 t! C+ |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.
- e3 y* ?. \1 Z+ h6 WAspirate the HEPES-BSS from the flask.
5 R0 p3 m! I0 r' |7 ICover the cells with 3 ml of Clonetics? T/E solution. Use cold (4?C) T/E solution for NHDF grown in FGM?. ) V# G* m0 Q6 h! {& Z4 b
Rock the flask to make sure all cells come into contact with the trypsin.
. X4 z6 I/ L5 ^& h, \$ P) y; Z, I/ ]Tighten the cap and begin monitoring the flask under the microscope.
" _+ s7 a, B0 {' p, w3 dContinue to examine the cell layer microscopically.
" ?* `: L4 m  s9 v$ ha. Allow the trypsinization to continue until 3 90% of the cells are rounded up." ?, _2 V" R" g/ n* {9 G

: `& z$ y& Z+ u! Q. |NOTE: 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.& d* q6 A4 q( M" C* c! @

4 {1 h& _: ~  c$ k! h$ k5 Mb. 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.
9 U  i# q! Y, p2 W1 F8 m; k. w
2 p& J& l/ t/ p# B/ V, c6 SNOTE: Don't try to get all cells to detach by rapping them severely. This action may damage the cells.! A$ J  l9 ?+ r- B  }9 Q
! d! V$ C, Z, ?2 @4 n
After cells are released, neutralize the trypsin in the flask with 3 ml of room temperature TNS.
3 P' ?0 F- S/ @5 m3 Y. s. K
7 m. k. K' v: f6 l1 Y- R1 r" xIf 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.7 e/ a5 o4 L- {- I, h; D) O- p* F
: D* B7 ?, i( G2 G# C
Quickly transfer the detached cells to a sterile 15 ml centrifuge tube. $ F; `' ?: }- j8 q- J' Q
Rinse the flask with a final 2 ml of HEPES-BSS to collect residual cells, and add this rinse to the centrifuge tube. 9 Z5 ?. t1 Q# }, s, n( t% w
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%. : u4 N" ?  b' E, s7 u- `
Centrifuge the harvested cells at 220 x g for 5 minutes to pellet the cells.
4 r- v. K* E+ d2 f6 t1 ~4 N0 L! {a. Aspirate most of the supernatant, except for 100-200 ml., a7 \5 ?, ]9 B
b. Flick the cryovial with your finger to loosen the pellet.
* [9 _3 x7 e  m+ \$ q. I
, L2 O- n* W  [Dilute the cells in 4-5 ml of growth medium and note the total volume of the diluted cell suspension.   h# C* {) L1 C2 C% h
During these procedures keep the cells in ice until they are plated.
# J! q8 r' [( B5 q* u$ j' }: s& `+ l) x( L, M: H% Q! A
To 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 c( `4 x5 S+ A$ [3 _5 m. W- V
% B" O5 H( w3 z' q+ ^2 ], B7 PCount 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.0 U# Q. l2 y+ ?3 I% g+ S
5 V  m' x- ]: b2 }
The cell suspension should contain between 250,000 to 1,000,000 cells/ml for greatest accuracy.  h$ s& i8 Y- n, m7 [* V" P

* x2 ?: d; U# T: _2 c0 DIf necessary, dilute the suspension with HEPES Buffered Saline Solution (HEPES-BSS) to achieve the desired "cells/ml" and re-count the cells.
+ G  ?) L* F  y) A9 ^* X1 u) A3 vAssess cell viability using Trypan Blue (see Appendix C). 7 g8 n" q8 M9 @
Use the following equation to determine the total number of viable cells:6 b) b9 m/ {5 N" e8 p5 J
* V( m' {/ e  x
Total # of Viable Cells = Total cell count x percent viability / 100+ s* r! E: @& h* V5 E! d
( [8 _' D  c. u6 W' q. P
Example: 1,000,000 cells x 60 / 100 = 600,000 viable cells 7 `: k; d# J; A: b) A

& {  p- U$ k# K3 cDetermine 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.* ]9 t1 ]+ ?) U" X5 L
! P* Q+ G& t8 E
NOTE: Recommended seeding density is 3500 cells/cm2 for NHDF and 2500 cells/cm2 for NHLF.9 L: ^- W: J. z: a2 L! y: ~

' Y! {% }" m8 ]6 hTotal # of flasks to inoculate = Total # of viable cells / Growth Area of Flask x Recommended Seeding Density& e0 {" n# e/ ?. @! Q

( v4 d5 E1 Z% FExample:600,000 viable cells / 75 cm2 x 3500 cells/cm2 = 2 T-75 flasks (rounded down to nearest whole number)
6 q6 Q) z- Z7 ~$ G( S1 o5 l6 E( `
% x$ v% I# p* E$ A! c2 v0 dUse the following equation to calculate the volume of cell suspension to seed into your flasks.; W0 B. \& j, M* Q

% N$ y- y0 h' O  i( ZSeeding volume = Total volume of diluted cell suspension / # of flasks as determined in step 18
% H! o- Q% r* V# F& C$ z! r
/ q3 C2 n3 |+ t$ wExample: 4.3 ml of diluted cell suspension / 2 T-75 flasks = 2.15 ml per T-75 flask0 _- Y7 F- ^! Q9 M: D
' A) a! q5 U9 H
Prepare flasks by labeling each flask with the passage number, strain number, cell type, and date. , {+ H- A+ {9 r/ I/ K
Carefully 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).
  L+ S2 {% w$ S) Q" y# n7 t9 x" {$ n: ~8 h2 n6 [  b
Example:15 ml growth medium for a 75 cm2 flask.; o" `% E, k' ~

5 V2 X3 R; n8 E. bAfter 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.
0 b- T5 G" }9 j3 n+ w# K- {After dispensing the cells, gently rock flask to promote even distribution.
; u8 u# V* S6 s1 V2 w0 @If 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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  \3 B# T, Z8 M( ~, ?( N
# r/ m/ H, {/ r+ E$ a
Assessing Yield and Viability# W8 E4 `5 q" v4 Z" V# i/ }
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:
. J, R- L$ F  w1 {  |' l5 N2 E
( t. m- s6 v! V7 X" q1. Study the sample chart below. It is a sample of high yield, high viability.
1 Q# Y: W0 I1 V2 b* @' V; I+ ]( A! D# q% L3 O; h4 |
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.4 A! j3 [" ^" N) J1 Z7 p

) w- ]2 ]. U5 Y& w* s$ L% N7 I) }b. Note the "solid dot" on the X axis or bottom line of the square. It indicates high viability, or more than 50% viability.6 e" c1 p8 p. r$ K/ B0 `

. _, p+ P/ c# g0 E2 Mc. 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.
0 r% u9 R$ R4 a& k
" @+ s8 d" m6 C3 J1 h. I  ]9 y2. Now, using the blank diagram below plot your cell yield and cell viability. Follow these steps:  R- r/ |  @; D* O+ T' p8 k, i

& F6 W: p7 _% q' Ba. Mark a (?) on the Y axis to indicate the total cell count of your culture./ Y0 c4 w; F6 t  n
7 o- w# m. b# z3 Q3 `; W
b Mark a (?) on the X axis to indicate the calculated percent viability of your culture.
* Q3 Y8 K! I& [& F3 W& _9 Z
/ e2 \) {/ v1 u/ r, `1 L; j' p/ H0 b1 ]$ A& S1 U
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.