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DRAFTCell LineProtocol
Version 1.0October 2010
Cover Art:MCF-10A phase contrast images were provided by Dr. Veronica Estrella (top; Moffitt PS-OC), and Drs. Denis Wirtz and Saumendra Bajpai (middle and bottom; JHU PS-OC).
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Cell Line Protocol
Contents
Overarching Goal ............................................................................................................................................................. 1
Proposed Solution............................................................................................................................................................ 3
Utility of Using Common Cancer Cell Lines as a Trans-Network Benchmark ............................................................. 3
Summary of Essential Standardization Elements ....................................................................................................... 5
Cell Lines ..................................................................................................................................................................... 5
Handling ....................................................................................................................................................................... 6
Minimum Annotation Data .......................................................................................................................................... 6
Appendix 1: Cell Lines ..................................................................................................................................................... 7
MCF-10A ...................................................................................................................................................................... 7
Characteristics of Interest .................................................................................................................................... 7
Applications.......................................................................................................................................................... 7
Cell Cycle Analysis ............................................................................................................................................. 10
Karyotyping......................................................................................................................................................... 10
Guidelines for Use .............................................................................................................................................. 12
MDA-MB-231 ............................................................................................................................................................ 13
Characteristics of Interest .................................................................................................................................. 13
Applications........................................................................................................................................................ 13
Cell Cycle Analysis ............................................................................................................................................. 16
Karyotyping......................................................................................................................................................... 17
Guidelines for Use .............................................................................................................................................. 20
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Cell Line Protocol
Appendix 2: Handling..................................................................................................................................................... 21
Subculturing/Passaging Adherent MCF-10A or MDA-MB-231 Cells ....................................................................... 21
Suspension ................................................................................................................................................................ 23
Subculturing in 3D ..................................................................................................................................................... 25
Thawing ..................................................................................................................................................................... 27
Freezing ...................................................................................................................................................................... 28
Mycoplasma Testing .................................................................................................................................................. 30
Appendix 3: Minimum Annotation Data ..................................................................................................................... 31
Appendix 4: Cell Culture Standards of Practice ....................................................................................................... 33
References ...................................................................................................................................................................... 43
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Cell Line Protocol
Overarching Goal
To test the feasibility of providing an initial common “benchmark” protocol that will be used by participating laboratories within the PS-OC Network to carry out and share the results of a set of pilot experiments to facilitate inter-PS-OC communications and accelerate the understanding of PS-OC specific technologies and techniques.
Before a common protocol can be discussed, it is important to prioritize a list of issues and constraints the solution must address:
• Timeframe: The PS-OC Steering Committee has charged that preliminary results from any protocol should begin being shared within the next 3 months via PS-OC intranet and presented formally by participating Center investigators at the annual PS-OC Network Investigators’ meeting in April 2010.
• Complexity of Protocol: Given the inherent potential of lab-to-lab variability, the developed protocol should include sufficient details so as to avoid data generated with mishandled cells. Bearing this in mind, the complexity of developing, optimizing, and carrying out the procedures will correlate heavily with the robustness of the cells and the defined scope. Providing one universal agonist to allow controlled perturbations is one of many options.
• Minimum Annotation Data: Independent of cell lines and technologies that are used by PS-OC participants for this exercise, the protocols should include a minimum set of annotations that are reported, including, but not limited to, passage number, subculture conditions, and images of the cells in culture and prior to experimentation.
• Adaptability for Future: With the eclectic nature of the PS-OC Network, it would be difficult to design a pilot protocol that encompasses all technologies and model systems. However, as the Network better understands each other’s strengths and technologies, the pilot protocol should be designed to be expandable and/or be able to serve as a jump-off point to develop separate protocols for more complex studies and model systems (mouse, clinical samples, etc.).
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Utility of Using Common Cancer Cell Lines as a Trans-Network BenchmarkWhile it may seem paradoxical that members of the PS-OC Network have been asked to blaze novel avenues of research using two cancer cell lines that have been in common use for several decades, it actually makes sense for these selected pilot experiments. The notion is that every group needs a common language in order to communicate, and applying the multitude of different technologies to one or two common cell lines in a pilot study will help initiate this process within the PS-OC network. Members of the network are studying various aspects of carcinogenesis that range from the initiation of cancer to metastasis of the tumor cells. Different “broken” or altered pathways typify each stage in this process. The PS-OC participants have reached a majority consensus that two human mammary cell lines will be used for the pilot studies: MCF-10A and MDA-MB-231. These two cell lines can be used for comparative studies of “pre-malignant” versus “malignant” signatures; however, this pair of cells is not amenable to making general conclusions about the nature of cancer. It is realized that no cell line would fill all criteria for being representative of any given cancer. The purpose of the chosen lines is not to represent the disease per se but to have commonly cultured standards for pilot experiments to showcase the various new technologies being developed or utilized by the multiple Centers.
Importantly, it is common knowledge that cell lines can vary in their properties as they are propagated in culture and that the culture conditions can select for different phenotypes [1]. To mitigate these problems, the cell lines indicated above will be cultured under standard conditions for the pilot experiments. A summary of potential standardization elements is included below (with the associated detailed descriptions included in Appendices).
Proposed Solution
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Cell Line Protocol
Summary of Essential Standardization Elements
Cell Lines [see Appendix 1] • MCF-10A:
– Growth medium: - DMEM:F12 (Invitrogen #11320-033),
supplemented with 5% horse serum (Invitrogen #16050-122), 0.5 μg/ml hydrocortisone (Sigma # H-0888), 20 ng/ml hEGF (Invitrogen #PHG0311 or PHG0313), 10 μg/ml insulin (Sigma I-0516), 100 ng/ml cholera toxin (Sigma C-8052), and 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen #15140148 or 15140122). Once reconstituted, the medium is good for 2 to 3 weeks at 4 °C.
– Serum: - Horse serum (Atlanta Biologicals cat
#S12195); Lot #J0116 has been put aside for common use by the PS-OCs.
– Subculture: - MCF-10A cells should be passaged at
a ratio of 1:5, roughly corresponding to 20,000 cells/cm2.
– Passage time: - MCF-10A cells should be passaged every
3-4 days. Cells should never be grown over 80% confluency.
– Maximum passage number: - MCF-10A cells in culture should not
exceed 10 passages from the time of receipt. Cells will “drift” after an excessive number of passages.
• MDA-MB-231:
– Growth medium: - DMEM (Invitrogen #11965-092)
supplemented with 10% fetal bovine serum (Atlanta Biologicals #S11095)).
– Serum: - Fetal bovine serum (Atlanta Biologicals cat
#S11095); Lot #G0079 has been put aside for the PS-OCs.
– Subculture: - MDA-MB-231 cells should be passaged
at a ratio of 1:8 to 1:10, roughly corresponding to 30,000 cells/cm2.
– Passage time: - MDA-MB-231 cells should be passaged
every 2-3 days. Cells should never be grown over 80% confluency.
– Maximum passage number: - MDA-MB-231 cells in culture should not
exceed 20 passages from the time of receipt. Cells will “drift” after an excessive number of passages.
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Handling [see Appendix 2]• Distribution:
– Cell lines should be provided by a single source that has the capacity to supply multiple aliquots in a consistent manner. Dr. Thea Tlsty has agreed to be the supplier of the designated cell lines.
– A single point of contact at each PS-OC should be responsible for the receipt and initial handling of cell line shipments.
• Subculturing/Passaging(page21):
– Cell lines should be initially expanded (i.e., passage 2) by the point of contact at each PS-OC.
– A minimum of 5 aliquots should be frozen and stored for each PS-OC investigator.
– Aliquots should be distributed to PS-OC investigators- Frozen if off site.- In a flask filled with Growth Medium if on
site.
• Thawing(page27):
– Frozen cells should be thawed quickly in a 37 °C water bath and plated IMMEDIATELY at a defined ratio or cell density (i.e., 1 vial per 10-cm plate or 10,000 cells/cm2).
• Freezing(page28):
– One-third of a subconfluent plate of cells (or approximately 1 million cells) should be frozen in 1 ml of Growth Medium containing 10% cell culture grade DMSO (sterile Hybri-maxTM; Sigma #D2650) per vial.
• MycoplasmaTesting(page30):
– Monitoring of cell cultures for mycoplasma contamination is by Bioluminescence Assay designed for testing cell-free medium (Lonza #LT07-118).
Minimum Annotation Data [see Appendix 3] • Phasecontrastimagesofcellsshouldbetaken:
– At passage 3 immediately before subculture.– Immediately prior to experimentation.
Literature pertinent to the specific cell line “batches” with which the PS-OCs will be working are as follows:
MDA-MB-231: Guise TA et al., J. Clin. Invest. 1996, 1544-1549 [2]
MCF-10A: Debnath J et al., Methods, 2003, 256-268 [3]
Important Note: 100 bottles each of the lots of fetal bovine serum and horse serum mentioned above have been placed on reserve for the PS-OCs. Arrangements have been made with the vendors so that each Center can purchase the serum directly without having to go through a centralized laboratory.
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Appendix 1: Cell Lines
MCF-10A:Brief History: MCF-10A is an adherent, immortalized, non-transformed human mammary epithelial cell line that arose spontaneously from MCF-10 cells. These cells were originally derived from a 36-year-old patient with fibrocystic changes [3]. In their original characterization, Soule et al. showed that MCF-10A cells lack tumorigenicity in nude mice, lack of anchorage-independent growth, as well as dependence on growth factors and hormones for proliferation and survival.
Characteristics of InterestMCF-10A cells arose spontaneously from the diploid MCF-10 cell line. MCF-10A cells cannot grow in soft agar [4] and, when injected in immunocompromised mice, are unable to form tumors (non-tumorigenic) [5]. However, the cells can grow in semisolid medium, and this has allowed for the development of a three-dimensional culture system where cells form structures that mimic the in vivo mammary tissue architecture. MCF-10As carry a near-diploid karyotype [6]; however, they have lost the p16 locus, an important regulator of cell cycle progression. The cells express normal p53, are estrogen and progesterone-receptor-negative [7-8], but express HER2 at low levels [9].
MCF-10As grow as a flat monolayer with cells that are heterogeneous in size and shape. The cell nucleus is relatively homogeneous in size and contains a prominent nucleolus. Based on microscopic characterization, the cells display luminal ductal cell characteristics [10].
ApplicationsMCF-10As are easy to manipulate and carry a nearly diploid karyotype. For this reason, these cells have been extensively used for the study of premalignant progression. Importantly, MCF-10A can be grown in three-dimensional cultures that recapitulate structures analogous to those found in vivo. Hence, MCF-10A have been extensively used in studying changes in cell growth, cell polarity, cell-to-cell adhesion, cell-to-cell junctions, and cell interactions with the extracellular matrix [11-14] associated with premalignant progression [7,15-21].
• Culture Conditions: MCF-10A cells are normally grown on DMEM/F12 supplemented with 5% horse serum, cholera toxin, hydrocortisone, insulin, and epidermal growth factor as listed in Table 1 [3].
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Growth Medium (with serum)
Resuspension Medium
3D-Assay Medium Overlay Medium Freeze Medium
DMEM/F12 DMEM/F12 DMEM/F12 DMEM/F12+2% Matrigel
Growth Medium (with serum)
5% Horse serum 20% Horse serum 2% Horse serum 2% Horse serum 10% (v/v) DMSO
0.5 µg/ml Hydrocortisone
0.5 µg/ml Hydrocortisone
0.5 µg/ml Hydrocortisone
20 ng/ml hEGF 5 ng/ml hEGF
10 µg/ml Insulin 10 µg/ml Insulin 10 µg/ml Insulin
100 ng/ml Cholera toxin
100 ng/ml Cholera toxin
100 ng/ml Cholera toxin
100 units/ml Penicillin, 100 µg/ml Streptomycin
100 units/ml Penicillin, 100 µg/ml Streptomycin
100 units/ml Penicillin, 100 µg/ml Streptomycin
100 units/ml Penicillin, 100 µg/ml Streptomycin
100 units/ml Penicillin, 100 µg/ml Streptomycin
Table 1. Recipes for serum-containing medium for growth in culture of MCF-10A cells are described in [3]. The original formulation was
published in [4]. A subcultivation ratio of 1:3 to 1:4 is recommended with media changes every 2 to 3 days. More details about growth
conditions for MCF-10A cells can be found at https://brugge.med.harvard.edu/protocols/ (Brugge Lab’s website). Important Note: Once
supplements (serum, growth factors, etc.) are added to the basal medium, the shelf life at 4 °C is about 4-6 weeks.
Cells are shown at various time points after initial seeding and at two magnifications (4x and 10x). Cell density shown at the 72 h time point is
optimal for passaging the cells. Cells shown at 120 h are overgrown.
MCF-10A Cell Cultures at Various Time Points After Seeding24 h 48 h 72 h 120 h
4x
10x
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The graph shown above is a typical growth curve for the MCF-10A cells. Note that the cells proliferate very nicely up to the 96 h time point,
whereas the cells that were overgrown on purpose (120 h time point) start to show a drop in proliferation due to high cell density and nutrient
deprivation. This reemphasizes the importance of passaging cells at an optimal 70%-80% density.
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
0 24 48 72 96 120
MCF-10A GROWTH CURVE
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MCF-10A Cell Cycle AnalysisA typical cell cycle analysis profile is shown above for the MCF-10A cell line. In contrast with nonproliferative cells, this profile shows a high percentage (57%) of cells in S phase (phase of DNA synthesis). A nonproliferative cell population would be characterized by a very small population in S phase (<2%-3%) with the remaining bulk of cells being within the Go/G1 or G2 phases.
MCF-10A KaryotypingKaryotyping was carried out by Molecular Diagnostic Services Inc. (San Diego, CA) under Project #100106-140.
Mitotic Arrest, Cell Harvest, and Slide PreparationCells were allowed to grow to 80% confluency. Mitotic division was arrested by treating the cell lines with 75 ng/mL Colcemid® for 18.5 hours. Following treatment, cells were harvested with Trypsin-EDTA, treated with a hypotonic solution, and then fixed in methanol/acetic acid. Metaphase spreads were prepared from treated and fixed cells and stained to observe chromosomal G bands. Multiple slides were prepared and 11 spreads were analyzed and karyotyped. A representative karyotype is presented below.
The cell line karyotype for this MCF-10A cell line sample is: 47,XX,+1,i(1)(q10),add(1)(q12),add(3)(p14),der(8)t(8;8)(p21;q21),der(9)(5qter->5q23:;3p25->3p21::9p2?2->9qter)[cp9]/96-97,idemx2[cp2]
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In the karyotype figure above, arrows indicate the regions of abnormality. Legends for the Table. 47,XX: female karyotype with one additional
chromosome (a normal karyotype would be 46,XX); +1: 3 copies of chromosome 1 with one copy being a fusion (isochromosome) between two
copies of the long arm of chromosome 1: (1q) i(1)(q10) and one copy showing additional material of unknown origin inserted at location 1q12
add(1)(q12); add(3)(p14): one copy of chromosome 3 displays additional material of unknown origin inserted at position 3p14; der(8)t(8;8)
(p21;q21): one copy of chromosome 8 is a derivative chromosome resulting from a translocation between 8p and 8q at locations 8p21 and
8q21; der(9)(5qter->5q23:;3p25->3p21::9p2?2->9qter): one copy of chromosome 9 is a derivative chromosome resulting from a three way
translocation between chromosome 5 (from the end of the long arm to 5p23), chromosome 3 (from 3p25 to 3p21 and chromosome 9 (from 9p2?2
[break point approximate+] to the end of the long arm of chromosome 9). So this contains all of the long arm and the short arm of chromosome
9 up to approximately 9p2.2. Note that there is some clonal heterogeneity, meaning that the karyotype indicates the abnormalities observed,
but not every cell karyotyped had every mentioned abnormality. Of 15 metaphases examined, 2 had approximate doublings of chromosome
number.
MCF-10A Cell Line Karyotyping
ResultsNumber of Metaphase Counted 11
Band Level 350-400Number of Metaphase Karyotyped 8
Number of Chromosomes 47Sex Female
Comments Some abnormalities foundKaryotype 47,XX,+1,i(1)(q10),add(1)(q12),add(3)(p14),der(8)t(8:8)(p21:q21),
der(9)(5qter->5q23:;3p25->3p21::9p2?2->9qter)[cp9]/96-97,idemx2[cp2]
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Cell Line Protocol
Description of the MCF-10A PS-OC Cell Line and Guidelines for Immediate UseEach PS-OC Center will be provided with 2 vials of frozen MCF-10A cells (5.3 x 106 cells/vial frozen on 12/25/09) at passage 13 (p13), passage doublings (PD) 2.97. Therefore, the cells underwent 2.97 passage doublings since they were obtained from Dr. Jayanta Debnath (UCSF). Given the growth curve of these cells (~1.5 PD/passage), their overall PD is estimated at ~20-22. Cells have been tested for the absence of Mycoplasma contamination by Bionique Testing Laboratories Inc. (Saranac Lake, NY). Each PS-OC was provided with 500 ml of MCF-10A medium READY TO USE (all additives including horse serum have been included). This medium should be used by 02/16/10.
Upon arrival, store the two vials of frozen cells in liquid nitrogen (or at -80 °C) and the medium at 4 °C. For expansion, keep 1 vial frozen as backup. Thaw out the other vial quickly in a water bath at 37 °C, mix it IMMEDIATELY with 45 ml of the medium we shipped to you, and dispense this suspension in a T125-T150 flask or split the suspension between three smaller T75 flasks (15 ml/suspension/flask). Seeding the vial in a single T75 flask would result in rapid overcrowding of the flask by the next day or the day after.
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MDA-MB-231:Brief History: MDA-MB-231 is an immortalized human metastatic cell line originally derived from a pleural effusion of a patient with metastatic breast carcinoma [22]. The patient presented with pericardial effusion and left pleural effusion and underwent a right-side mastectomy. Treatments included oophrectomy, 5-fluorouracil, prednisone, and a triple chemotherapeutic cocktail of cyclophosphamide, doxorubicin, and methotrexate. The present cell line is derived from cells collected 3 weeks after chemotherapy. [22] MDA-MD-231 cells have been characterized as highly malignant, invasive and aggressive. Therefore, these cells can be used as a standard “cancer” cell line.
Characteristics of InterestMDA-MB-231 cells injected into immunocompromised mice exhibit highly malignant properties and are capable of forming tumors [23]. In 2-dimensional tissue culture, these cells are spindle-shaped and grow as an adherent monolayer [24]. In 3-dimensional culture cells form stellate projections associated with high levels of invasiveness [25]. Cytological and expression analyses have shown mesenchymal-like characteristics associated with aggressive cancers in these cells.
Disruptions in genome integrity lead to gross chromosomal rearrangements such as translocations, deletions, and amplifications. Although cytogenetic observations can vary slightly from one laboratory to another, a lack of normal chromosome 8 and chromosome 15 have been commonly reported [26,27]. Of particular interest is the presence of a single, mutant copy of TP53, which encodes the tumor suppressor p53, and deletion of both copies of CDKN2A, whose product p16 is important in regulating cell cycle progression [28,29].
Importantly, in addition to the characteristics listed above, this cell line is estrogen receptor negative (ER-), progesterone receptor negative (PR-), and human epidermal growth factor negative (HER2-). Cancers with this triple negative profile are aggressive and difficult to treat.
ApplicationsMDA-MB-231 cells share many features found in cancers associated with poor prognosis. Therefore, this cell line is an attractive model in which to study the characteristics that confer carcinogenic properties in highly malignant cancers. Examples of recent work capitalize on some of these phenotypes. As a model for metastasis, this cell line was transfected with cell-cell adhesion factors in an attempt to restrain its motility by one group. [30] In another group, the cell line’s resistance to chemotherapy was useful in dissecting pathways that could be modified to improve sensitivity to previously ineffective treatments. [31] In a third, factors that modulated apoptosis in MDA-MB-231 were examined as potential targets for cancer therapy. [32]
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Growth Medium (with serum)
Resuspension Medium
3D-Assay Medium Overlay Medium Freeze Medium
DMEM DMEM DMEM DMEM+2% Matrigel
Growth Medium (with serum)
10% FBS 10% FBS 10% FBS 10% FBS 10% (v/v) DMSO
Table 2. Culture Conditions: MDA-MB-231 cells are normally grown on DMEM with high glucose supplemented with 10% fetal bovine serum
and L-glu. A subcultivation ratio of 1:3 to 1:4 is recommended with media changes every 2 to 3 days. These cells can be grown either in
monolayer or in suspension, thus allowing the same cell line to be used to showcase assays on adherent cells and cell suspensions. Important
Note: Once supplements (serum, growth factors, etc.) are added to the basal medium, the shelf life at 4 °C is about 4-6 weeks.
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Cell Line Protocol
MDA-MB-231 cells are shown at various time points after initial seeding and at two magnifications (4x and 10x). Cell density shown at the 96 h
time point is optimal for passaging the cells. Cells shown at 144 h are overgrown.
The curve shown above is a typical growth curve for the MDA-MB-231 cells. Note that the cells proliferate very nicely up to the 96 h time point
whereas the cells that have been overgrown on purpose (120 h time point) start to show a drop in proliferation due to high cell density and
nutrient deprivation. This reemphasizes the importance of passaging cells at an optimal 70%-80% density.
MDA-MB-231 GROWTH CURVE
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MDA-MB-231 Cell Cycle AnalysisA typical cell cycle analysis profile is shown below for the MDA-MB-231 cell line. In contrast with nonproliferative cells, this profile shows a high percentage (22%) of cells in S phase (Phase of DNA synthesis). A nonproliferative cell population would be characterized by a very small population in S-phase (<2-3%), with the remaining bulk of cells being within the Go/G1 or G2 phases.
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Cell Line Protocol
MDA-MB-231 KaryotypingKaryotyping was carried out by Molecular Diagnostic Services Inc. (San Diego, CA) under Project #100203-140.
Mitotic Arrest, Cell Harvest, and Slide Preparation:Cells were grown to 80% confluency. Mitotic division was arrested by treating the cell lines with 75 ng/mL Colcemid® for 18.5 hours. Following treatment, cells were harvested with Trypsin-EDTA, treated with a hypotonic solution, and then fixed in methanol/acetic acid. Metaphase spreads were prepared from treated and fixed cells and stained to observe chromosomal G bands. Multiple slides were prepared and multiple spreads were analyzed and karyotyped. A representative karyotype is presented below.
The cell line karyotype for this MDA-MB-231 cell line sample is:
60-62,XX,-X,+1,add(1)(p13),add(1)(p32),add(2)(p12),+3, add(3)(q12),+4,+5,add(5)(p15),+6,+6,add(6)(q12),add(6)(q13),+add(6)(q13),+7,add(8)(q22),del(9)(p21)X2,del(9)(p22),+10,+11,del(12)(q21),-14,add(15)(p11.2),add(15)(p11.2),+17,+18,add(18)(q21),+19,add(19)(p13.3),+20,add(20)(p12),add(21)(p11.2),add(21)(p11.2)+2-7mar[cp15]
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Cell Line Protocol
In the karyotype figure above, arrows indicate the regions of abnormality. The circle indicates that in some clones the X chromosome is missing
(XO mosaic). Legends for the Table. 60-62,XX: female karyotype with 60 to 62 chromosomes (a normal karyotype would be 46,XX); +1: 3 copies
of chromosome 1 with one copy showing additional material of unknown origin inserted at location 1p13 add(1)(p13) and 1p32 add(1)(p32);
add(2)(p12): one copy of chromosome 2 displays additional material of unknown origin inserted at position 2p12; +3: 3 copies of chromosome
3 with one copy showing additional material of unknown origin inserted at location 3q12 add(3)(q12); +4: 3 copies of chromosome 4; +5: 3
copies of chromosome 5 with one copy showing additional material of unknown origin inserted at location 5p15 add(5)(p15); +6, +6: 5 copies
of chromosome 6 with one copy showing additional material of unknown origin inserted at location 6q12 add(6)(q12), and the two other copies
showing additional material of unknown origin inserted at location 6q13 add(6)(q13); +7: some cells have an additional chromosome7; each
copy of chromosome 8 shows additional material of unknown origin inserted at location 8q22 add(8)(q22); 3 copies of chromosome 9 with
two copies showing partial deletions of the short arm starting at 9p21 del(9)(p21)X2 and one copy showing a partial deletion of the short
arm starting at 9p22 del(9)(p22); +10: 3 copies of chromosome 10; +11: 3 copies of chromosome 11; one copy of chromosome 12 showing a
partial deletion of the long arm starting at 12q21 del(12)(q21); -14: one copy of chromosome 14 is missing in some cells (it is present in the cell
presented here); both copies of chromosome 15 show additional material of unknown origin inserted at location 15p11.2 add(15)(p11.2); +17: 3
MDA-MB-231 Cell Line Karyotyping
ResultsNumber of Metaphase Counted 15
Band Level 300-400Number of Metaphase Karyotyped 15
Number of Chromosomes 60-62Sex Female
Comments Multiple abnormalities foundKaryotype 60-62,XX,-X,+1,add(1)(p13),add(1)(p32),add(2)(p12),+3,
add(3)(q12),+4,+5,add(5)(p15),+6,+6,add(6)(q12),add(6)(q13),+add(6)(q13),+7,add(8)(q22),del(9)(p21)X2,del(9)(p22),+10,+11,del(12)(q21),-14,add(15)(p11.2),add(15)(p11.2),+17,+18,add(18)(q21),+19,add(19)(p13.3),+20,add(20)(p12),add(21)(p11.2),add(21)(p11.2)+2-7mar[cp15]
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copies of chromosome 17; +18: 3 copies of chromosome 18 with one copy showing additional material of unknown origin inserted at location
18q21 add(18)(q21); +19: 3 copies of chromosome 19 with one copy showing additional material of unknown origin inserted at location 19p13.3
add(19)(p13.3); +20: 3 copies of chromosome 20 with one copy showing additional material of unknown origin inserted at location 20p12
add(20)(p12); one copy of chromosome 21 showing additional material of unknown origin inserted at location 21p11.2 add(21)(p11.2). Cells
contain anywhere from 2 to 7 marker chromosomes of unknown origin +2-7mar[cp15]. Note that there is some clonal heterogeneity, meaning
that the karyotype indicates the abnormalities observed, but not every cell karyotyped had every mentioned abnormality.
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Cell Line Protocol
Description of the MDA-MB-231 PS-OC Cell Line and Guidelines for Immediate UseEach PS-OC center will be provided with 2 vials of frozen MDA-MB-231 cells (1e6 cells/vial frozen on 02/08/10) at passage 32 (p32), PD 1.2. This means that the cells underwent 1.2 passage doublings since we got the cells from ATCC. Given the growth curve of these cells (~1 PD/passage), their overall PD is estimated at ~30-32. We also provide each PS-OC center with 500 ml of MDA-MB231 medium READY TO USE (fetal bovine serum has been included). This medium should be used within 4-6 weeks after delivery. Cells have been tested for the absence of Mycoplasma contamination by Bionique Testing Laboratories Inc. (Saranac Lake, NY).
Upon arrival, store the two vials of frozen cells in liquid nitrogen (or at -80 °C) and the medium at 4 °C. For expansion, keep 1 vial frozen as backup. Thaw out the other vial quickly in a water bath at 37 °C, mix it IMMEDIATELY with 15 ml of the medium we shipped to you, and dispense this suspension in a T75 flask. Seeding the vial in a single T25 flask would result in rapid overcrowding of the flask within 2-3 days.
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Cell Line Protocol
Appendix 2: Handling
Subculturing/Passaging Adherent MCF-10A or MDA-MB-231 Cells
Materials:• Growthmedium• Resuspensionmedium• 10-cm2 polystyrene plates (Nunc/ThermoFisher Scientific #150679 or 172958)• 15-mlpolypropyleneconicaltubes• Phosphate-bufferedsaline(PBS)• Trypsin/EDTA(0.05%trypsin(0.5g/L);0.53mMEDTA(0.2g/L);6.9mMNaHCO3 (0.58g/L))• 37°Cwaterbath
Method:Note: Be sure to keep track of the passage number or population doubling number. With each subculture, the passage number increases by 1. In contrast, with each subculture, population doubling number increases in relationship to the dilution at which the cells are plated. Depending on the cell line, there is a specific passage number (or population doubling number) that should not be exceeded.
Subculturing by Passage Number:1. Place all medium and trypsin in the 37 °C water bath for at least 15 min prior to beginning this procedure.2. Using a Pasteur pipette attached to the open end of the vacuum hose, aspirate medium from the plate.3. Add 10.0 ml of PBS to the plate to rinse. 4. Aspirate the PBS and add 2.0 ml of Trypsin/EDTA.5. Incubate the plate at 37 °C in a 5% CO2 humidified incubator for 3 min (MDA-MB-231) or 15 min (MCF-10A). This
incubation time is highly cell type-dependent. Gently tap the plate to help dislodge the cells. Check the plate under a microscope to ensure all cells have dissociated from the plate before proceeding to the next step.
6. Add 2.0 ml of Resuspension Medium to the plate. Pipette medium a few times to break up any cell clumps.7. Using a pipette, transfer the cells to a 15-ml conical tube. Add an additional 1.0 ml of Resuspension Medium to
rinse the plate and transfer to the 15-ml conical tube.8. Centrifuge cells at 900 rpm (160xg) at room temperature for 5 min.9. Carefully aspirate the medium away from the cell pellet. 10. Using a P-1000 pipetman, resuspend the cells in 1.0 ml of Growth Medium. Pipette repeatedly and gently to
obtain a uniform single cell suspension without damaging the cells. 11. Add an additional 4.0 ml of Growth Medium to the tube and pipette to mix.
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12. To each 10-cm plate onto which cells will be seeded, add 10.0 ml Growth Medium. To this, add 1.0 ml of the cell suspension for a 1:5 dilution. Cells seeded at passage ratios of 1:5 and 1:6 will become confluent in roughly 3 and 4 days, respectively.
13. Maintain these cell cultures at 37 °C in a 5% CO2 humidified incubator.14. Change the medium every 2-3 days. To do this, aspirate the old medium and add 10.0 ml fresh Growth Medium
to the 10-cm plate. Cells should be subcultured (passaged) every 3-4 days. Cells should never be grown over 80% confluency during expansion.
Subculturing by Population Doubling:1. Handle cells as described in steps 1-11 described above for subculturing by passage number.2. Count cells using a hemacytometer or an automated cell counter. Transfer a given number of cells (for example,
5 x105 cells) to a fresh plate that contains media. Swirl the plate to disperse cells evenly and place in incubator to allow cells to attach and grow. Continue with steps 13 and 14 described above. Since one calculates the number of cells that are placed in the fresh plate and counts the number of cells removed from the plate after a given amount of time, it allows one to obtain the information needed to calculate the increase in cell number per unit time. This is an accurate method of documenting cell kinetics and determining the number of times the population doubles per passage. Population doubling (PD) is calculated as follows: PD = log (number of cells recovered/number of cells seeded)/log 2. Example: you seed 1 million cells. 4 days later you trypsinize cells to passage them and count 4 million cells; the increase in passage doubling of this cell culture is log (4/1)/log 2 = 0.6/0.3 = 2. In other words, this culture has undergone 1 passage (you passage the cells one time) but 2 passage doublings (cells divided twice).
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Subculturing/Passaging Cells in SuspensionThis type of cell culture is a complement (or alternative) to 3D cultures in Matrigel. Cells are grown on very low attachment plasticware which precludes their adhesion. Instead of forming 2D adherent monolayers, cells form 3D spheres [33]. Ultra low attachment surface results from a unique covalently bonded hydrogel surface that is hydrophilic and neutrally charged. It minimizes cell attachment, protein absorption, and enzyme activation. The surface is non-cytotoxic, biologically inert, and nondegradable. The protocol below is for 6 well plates and has been optimized for MCF-10A cells [33]. MDA-MB-231 cells are very resilient and are expected to grow well in this same medium. Other ultra low attachment surface dishes or multiwell plates are available: Fisher Scientific 05-539-100 (Corning #3261) for 60-mm dishes, 05-539-101 (Corning #3262) for 100-mm dishes, 07-200-601 (Corning #3471) for 6 well clusters, 07-200-602 (Corning #3473) for 24 well clusters, 07-200-603 (Corning #3474) for 96 well clusters. Adjust volumes accordingly if other vessels are used.
Materials:• MEBMBasalMedium,(Lonza,Walkersville,#CC3151)supplementedwith:
– B27 (Invitrogen #17504044)– 20 ng/ml EGF (Invitrogen #PHG0311 or PHG0313)– 1 μg/ml hydrocortisone (Sigma # H-0888)– 5 μg/ml insulin (Sigma #I-0516)– 5 μg/ml β-mercaptoethanol (Sigma #M7522)
• Resuspensionmedium(fromTables1and2)• Hemocytometerorautomatedcellcounter• 15-mlpolypropyleneconicaltubes• Sixwellultralowattachmentplate(Corning#3471;FisherScientific#07-200-601)• Phosphate-bufferedsaline(PBS)• Trypsin/EDTA(0.05%trypsin(0.5g/L);0.53mMEDTA(0.2g/L);6.9mMNaHCO3 (0.58g/L))• 37°Cwaterbath
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Method:Note: Be sure to keep track of the passage number. With each subculture, the passage number increases by 1. Depending on the cell line, there is a specific passage number that should not be exceeded (refer to “Maximum passage number” on page 5).
1. Follow the steps described in the previous section (Subculturing/Passaging Adherent Cells) up to step 7.2. Count cells using a hemacytometer or an automated cell counter. Calculate the number for cells needed for
each cell line at (5,000 cells/ml) in MEBM media supplemented with B27, 20 ng/ml EGF, 1 μg/ml hydrocortisone, 5 μg/ml insulin, 5 μg/ml β-mercaptoethanol.
3. Centrifuge cells at 900 rpm (160xg) at room temperature for 5 min.4. Carefully aspirate the medium. 5. Resuspend the cells in MEBM with all supplements at 5,000 cells/ml.6. Plate 2 ml of resuspended cells (10,000 cells) per well on a six well ultra low attachment plate.7. Maintain these cell cultures at 37 °C in a 5% CO2 humidified incubator.8. Change the medium (1 ml) every 2-3 days. IMPORTANT NOTE: Because cell spheres are non-adherent, they can
be very easily aspirated (and therefore lost) upon medium change. To avoid this, it is recommended: (a) to gently centrifuge the vessel to spin down cell spheres (5 min, 900 rpm [160xg]); and (b) to aspirate the old medium with a pipetman instead of a vacuum line to limit and control suction forces. Add 10.0 ml fresh Growth Medium to the 10-cm plate. Alternatively fresh medium can be added without removing the old medium or removing only part of it (this may be a good option at the beginning of the growth of the spheres).
9. After 7-10 days, MCF-10A cells should form mammospheres whereas MDA-MB-231 should organize in less structured cell clusters.
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Subculturing/Passaging Cells in a 3D Structure
Materials:• Assaymedium(fromTables1and2)• Resuspensionmedium(fromTables1and2)• Overlaymedium(fromTables1and2)• BDFalcon8-wellchamberSlideor8-wellglasschamberslides[Lab-Tek(Nunc)]• Shallowicebucketfilledwithice• Chilledpipettes• Matrigel(BDBisociences).IMPORTANTNOTE:Matrigelstockiskeptfrozenat-80°Candshouldbethawed
ON ICE just prior to use. Matrigel stays in liquid form at 0 °C to 4 °C. Thawing Matrigel at room temperature will result in gel formation, making it useless. In addition, any vessel (plate, chamber slide, pipette, etc.) used to aspirate, transfer, or lay Matrigel for 3D cell cultures should be pre-chilled prior to use; otherwise, Matrigel would very quickly form a gel prior to being spread on a dish or during transfer in a pipette).
• 15-mlpolypropyleneconicaltubes• Phosphate-bufferedsaline(PBS)• Trypsin/EDTA(0.05%trypsin(0.5g/L);0.53mMEDTA(0.2g/L);6.9mMNaHCO3 (0.58g/L))• 37°Cwaterbath• Hemocytometerorautomatedcellcounter
Method:1. Thaw Matrigel on ice at 4 °C overnight. Matrigel should be handled on ice at all times.2. Place an 8-well glass chamber slide on ice.3. Add 45 μl of Matrigel to each well of A PRE-CHILLED 8-well chamber slide (BD Falcon CultureSlide) and spread
evenly in the well. Take care not to generate air bubbles or touch the pipette tip to the edge of the well (this will form a high meniscus on the border). Place the slides in a cell culture incubator to allow the Matrigel to gel completely (takes 30-40 min). This step will require small-scale practice prior to being performed successfully in actual experiments. Matrigel is tricky to handle and expensive !!!!!
4. Regarding the cells, follow the protocol as described for the adherent cell up to step 5 and resuspend in 1 ml of resuspension medium (Please see Tables 1 and 2 above). Add an additional 9.0 ml of resuspension medium to the tube to fully quench trypsin.
5. Count cells using a hemacytometer.6. Spin the cells (from step 3) at 900 rpm (160xg) for 3 min. Aspirate the supernatant.
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7. Resuspend the cells in Assay Medium (Please see Tables 1 and 2 above) to achieve 25,000 cells/ml according to the cell count in step 5.
6. Prepare a 2X concentrated stock solution of Overlay Media (please see Tables 1 and 2 above) for the total number of wells + 1 (400 μl overlay media/well).
7. Transfer an appropriate amount of 2x overlay media (n + 1 wells/cell line x 400 μl) to a fresh tube and add an equal volume of cell suspension (to reach a final 1x overlay medium concentration).
8. Plate 400 μl of the above mixture to a well on top of the solidified Matrigel (from step 3). This step should be performed slowly so as not to dislodge the Matrigel layer. This should result in 5,000 cells/well in Overlay media (please see above table 1 and 2).
9. Allow the cells to grow in the incubator for approximately 10-15 days. The cells should be fed with fresh Overlay media every 4 days. The day the assay is set up is day 0, then feed cells on days 4, 8, 12, 16, etc. The cells will form spheres by about day 5-6 and will start forming hollow lumen structures by day 7-8 for MCF-10A cells or cell clumps for MDA-MB-231 cells.
Very helpful tips about 3D culture techniques can also be found in: Lee G et al. [34].
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Thawing Cells
Materials:• Basemedia(e.g.,DMEM/F12orDMEM)• GrowthMedium(seeTables1and2)• Icebucketfilledwithdryice• 37°Cwaterbath• 10-cm2 polystyrene plates (Nunc/ThermoFisher Scientific #150679 or 172958)• 15-mlpolypropyleneconicaltubes
Method:1. Place Growth Medium in the 37 °C water bath for at least 15 min prior to beginning this procedure.2. Retrieve a vial of frozen cells from liquid nitrogen and place on dry ice.3. Label plates to be seeded with the cells. Be sure to note the passage number. 4. Place the vial containing frozen cells in a 37 °C water bath for 2 min. Check the vial to see if the suspension
is thawed. If not, check every minute thereafter. Once cells are thawed (should not take more than 5 min), immediately remove the vial from water bath and process them as follows. IF NOT PLACED IN CULTURE QUICKLY THE VIABILITY OF THAWED CELLS WILL DECREASE DRAMATICALLY.
5. Add 10.0 ml of DMEM/F12 to a 15-ml conical tube.6. To the DMEM/F12, add the 1.0 ml thawed cell suspension.7. Centrifuge at 900 rpm (160xg) at room temperature for 5 min.8. Carefully aspirate the medium away from the cell pellet. 9. Using a P-1000 pipetman, resuspend the cells in 1.0 ml of Growth Medium. Pipette repeatedly and gently to
obtain a uniform single cell suspension without damaging the cells. 10. Add an additional 4.0 ml of Growth Medium to the tube and pipette to mix. 11. To the 10 cm2 plate where cells will be seeded, add 5.0 ml Growth Medium. To this, add the 5.0 ml of cell
suspension (final medium volume = 10.0 ml).12. Maintain these cell cultures at 37 °C in a 5% CO2 incubator. 13. Change medium the day after thawing/plating to remove any floating dead cells.14. Cells may need to be subcultured as early as 2 days post-plating.
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Freezing Cells
Materials:• FreezeMedium(seeTable1and2)• Icebucketfilledwithice• Cellfreezingvials(2.0ml)• Cellfreezingcontainer[e.g.,Mr.Frosty(Nalgene)]• -80°Cfreezer• Liquidnitrogentank
Method:1. Place Freezing Medium in ice bucket filled with ice.2. Label vials for freezing, including information about the passage number.3. Follow the “Subculture by passage number” method to #9. 4. Using a P-1000 pipetman, resuspend the cells in 2.0 ml of Freeze Medium. Pipette repeatedly and gently to
obtain a uniform single cell suspension.5. To each of two freezing vials, transfer 1.0 ml of the cell suspension. 6. Place vials on ice for 5 min.7. Transfer vials to a cell freezing container (optimally in a “Mr. Frosty” container which allows progressive freezing
of the cells or alternatively in a Styrofoam box) and place in a -80 °C freezer for at least 24 h.8. Transfer frozen cells to liquid nitrogen within 1 week (the day after the freezing is optimal).9. Cells can be stored indefinitely in liquid nitrogen (-196 °C).
Shipment:1. It is advised that cells should be shipped on Mondays, Tuesdays, or Wednesdays to minimize the risk for the
package to sit on a loading dock over the weekend.2. Notify the receiver to expect a package the following day, and ensure that they will be available for receipt of
the package.3. Arrange for a FedEx pickup and overnight delivery. 4. Fill a styrofoam box halfway with dry ice.5. Place the vial(s) of frozen cells along with an information sheet about the cells in a plastic bag.6. Place the bag of vials and the information sheet in the dry ice.7. Fill the remainder of the box with dry ice.
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8. Seal the lid of the Styrofoam box securely with packing tape. 9. Place the FedEx label on the box.10. Ensure that the box is picked up by FedEx and goes out for overnight delivery.
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Mycoplasma TestingMycoplasma testing has been performed on the cells that have been sent to each PS-OC group. If cells begin to alter their appearance, one may wish to perform mycoplasma testing in the recipient’s laboratory. This can be done on a frozen aliquot of cells after initially thawing the cells and before the first passage.
Materials:• MycoAlert® Mycoplasma Detection Kit (Lonza #LT07-118)• Luminometer
Method:1. Follow the manufacturer’s recommended protocol supplied with the kit.
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Appendix 3: Minimum Annotation Data
Independent of cell lines and technologies that are used by PS-OC participants for this exercise, the protocols should include a minimum set of annotations that are reported. These annotations include, but are not limited to, passage number, subculture conditions, and images of the cells in culture and prior to experimentation.
It is strongly encouraged that investigators share images of their cells in culture on the PS-OC Intranet prior to the Network Investigators’ Meeting April 2010. That way investigators can conduct early comparisons of cell aliquots after Center-specific culturing.
For example, at least a panel of two images should be shown:
Phase contrast image of passage 3 cells in culture prior to subculturing/passaging
Phase contrast image of cells in culture prior to experimentation
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This section is intended for people who have no or little experience with cell culture and/or limited access to and interaction with facilities routinely performing cell culture. Institutional training sessions will extensively cover biosafety requirements and cell culture standards of practice. The following summarizes important guidelines that ensure good practice and also highlights common mistakes that often lead to culture bacterial and/or fungal contamination. Always wear labcoats dedicated to cell culture. Wearing eye protection devices is highly recommended (mandatory in some institutions). DO NOT PLAN A DAY ALTERNATING WORK WITH CELLS AND BACTERIA.
Room:• Thecellcultureroomshouldbecheckeddailyforsuppliesandcleanliness.• Biosafetycabinets(oftenreferredtoascellculturehoods)shouldbeinspectedyearly.• Biohazardouswastehardcontainers(fordisposalofglasspipettes),biohazardouswastebags(forplastic
pipettes and biohazardous material) and biohazardous liquid waste should be disposed of on a regular basis. NEVER DISPOSE OF GLASS VESSELS OR PIPETTES IN A SOFT BIOHAZARDOUS BAG.
• Priortouse,biosafetycabinetsshouldbesterilizedbyturningonUVlight(frontslidingdoorclosed)andthenlifting the front sliding door and turning on the blower for at least 20 minutes.
Incubators:• TheMCF-10AandMDA-MB-231celllinesaregrown(likemostcelllines)at37°Cinahumidifiedatmosphere
and in the presence of 5% CO2. • Followmanufacturer’sguidelinesforincubatorinstallationandmaintenance.• Surfaces(inparticulartheinternalglassdoorandshelves)shouldberegularlysprayedwithethanol.Itis
recommended to autoclave internal removable parts (sides, shelves, and shelf supports) every 3 months.• Thelevelofwaterinthepanatthebottomoftheincubatorshouldbecheckedregularlyandwatershouldbe
replaced every week. Additionally, water-jacketed incubators need regular addition of water within the walls of the incubator itself. An alarm in the front of the incubator will warn you if the level is too low. ALWAYS USE STERILIZED DISTILLED WATER, NOT DEIONIZED WATER.
• Immediatelycleanupanyspillofmedium.
Appendix 4: Cell Culture Standards of Practice
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Water Bath:• Thelevelofwaterinthewaterbathshouldbecheckedregularly.Asdescribedabove,cleanupregularlyand
replace water (distilled water only) once a month. The addition of a reagent such as “CLEAR BATH” algaecide will avoid fungal contamination.
Plasticware/Glassware:• Thetypeofplasticshouldbechosenaccordingtothetypeofexperiments.Cellcultureplasticwareiscoatedto
enhance cell adhesion. Special low-adhesion plasticware should be used to carry out experiments with cells in suspension.
• ExceptforPasteurpipettesusedtoaspiratemediumoutofcellcultureflasksordishes,mostlaboratorieshavebanned the use of recycled (washed and sterilized) glass pipettes for cell culture. Single-use plastic pipettes come in various sizes (1, 2, 5, 10, 25, and 50 ml). Some of these pipettes are available as aspirating pipettes (without cotton plugs in the top section of the pipette) used instead of Pasteur pipettes. This alternative is recommended to avoid the most common injuries reported in tissue culture settings: crushing a Pasteur pipette while connecting it to the aspiration tubing or shattered glass while hitting the pipette on a surface as a result of distraction. To avoid the first type of incident, a good alternative is to plug a P1000 pipette tip (cut at the tip to widen its opening) on the tubing.
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• Greatcareshouldbegiventokeepvesselssterile,especiallywhenpacksofdishesorflaskshavebeenopen.• Plasticpipettesusedforcellcultureshouldalwaysbepurchasedindividuallywrapped(notinbulkbags).• Althoughmoreexpensive,tissuecultureflaskscanbepurchasedwithventedlids,allowingcellstogrowin
tightly capped flasks. Flasks with nonvented lids require the lids to be slightly loosened when flasks are placed in the incubator (this can potentially be a source of bacterial/fungal contamination).
• Considerpurchasingplasticwarefromvendorsatadiscountedpricethatisnotnormallyofferedbythemanufacturer.
Media:• Basalmediumisstoredat4°Candisusuallygoodfor6-12monthspriortoreconstitution.• Mediashouldbereconstituted(additionofvariouscomponentstobasalmediumsuchasserum,growthfactors,
and hormones) for short-term storage. Once reconstituted, a medium shelf life is 4-6 weeks at 4 °C (do not re-freeze). ALWAYS WRITE THE DATE OF SUPPLEMENT ADDITION ON THE BOTTLE.
• Trypsin,whichisusedtodetachcells,aswellasserum,hormones,andgrowthfactorsarekeptfrozenforlong-term storage. Trypsin usually comes as 0.05% and 0.25% solutions. The 0.25% solution is harsher on the cells. It is recommended to use 0.05% whenever possible.
• Allreagentsshouldbemixed(inverted)priortouse,particularlyfrozenitemsafterthawing.
Cell Culture Practices:• Stepsaredescribedabovetopreparethebiosafetycabinetpriortouse(seefourthbulletinthe“Room”section).• Oncereadyforuse,spraywith70%ethanoltheoutsideandinsideofthefrontslidingdoor,andtheworking
surface in the hood.
• Anyitemintroducedinthesterileworkingspace(bottlesofmedia,racks,flasks,etc.)shouldalsobesprayedwith 70% ethanol prior to being introduced in the sterile environment. SPRAY YOUR GLOVES REGULARLY.
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• Itiscommontoleaveinthehoodarackforsterileindividuallywrappedpipettesandboxesoftips.Thisisoptional.
• Refertoguidelinesforpassagingcellsinaprevioussection.Theguidelinesbelowareonlysafepratices:• Unwrappingapipette:Peeloffbothsidesofthewrappingstartingfromthetopofthepipette,notfromitstip.
Make sure to store the pipettes with tops up or facing you. You will naturally pick the pipette and open it at the top not the tip. CONTAMINATION OF THE LOWER PART OF A PIPETTE IS THE BEST WAY TO CONTAMINATE YOUR CELL CULTURE.
The panels above illustrate proper unwrapping of a pipette (the pipette is opened from the top end).
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The panel above shows WHAT YOU SHOULD NOT DO (the pipette is being opened from the tip end).
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Pipetting Medium Out of a Bottle and Transferring It in a Flask:• Keepthetimeduringwhichabottleoraflaskisopentoaminimum.• Alwaysaimforthecenteroftheopeningofthebottleoroftheflask(seebelow).Touchingtheneckofthebottle
or of the flask will increase the risk of contamination. • ALWAYSMOVEYOURHANDAROUNDANOPENVESSELNEVERABOVEIT.Manycontaminationsoccurbecause
unprotected parts of the arm (neither covered by the labcoat nor the gloves) such as the wrist are in contact with open vessels.
• Guidelinesforvolumerangesofcellculturemediumtodispenseincommoncellculturevessels:– Flasks: T150 (40-45 ml), T75 (15-20 ml), T25 (5-7 ml)– Dishes: P150 (30-35 ml), P100 (18-20 ml), P60 (7-8 ml), P35 (2-3 ml)– Clusters (volume/well): 6 wells (3-4 ml), 12 wells (1.5-2 ml), 24 wells (0.5-1 ml), 48 wells (250-500 μl),
96 wells (125-250 μl) Note: When cells are at a higher density, use the larger volumes of medium that are recommended. At
higher cell densities, the medium will be depleted of nutrients more rapidly than when cell density is low.
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• Ifinadvertentlyapipette(evenunused)touchesyourlabcoat,glove,skin(oranythingotherthanthesterilemediainside the bottle; see examples below), discard it right away and do not use it to pipette medium or to transfer cells.
Freezing and Thawing Cells:Cells should be frozen slowly by placing the cryogenic vial in a pre-cooled “Mr. Frosty” freezing container (Nalgene #5100-0001) or alternatively by placing the cryogenic vial in a Styrofoam rack placed itself in a Styrofoam box in a -80 °C freezer. For long-term storage, vials are transferred the following day to a liquid nitrogen container. DO NOT PUT THE VIAL OF CELLS TO BE FROZEN IMMEDIATELY IN LIQUID NITROGEN.
In contrast, cells have to be thawed very quickly in a 37 °C water bath and placed IMMEDIATELY in culture. Therefore when thawing a vial of frozen cells, the recipient dish or flask should be ready with cell culture medium already dispensed within it. The thawed cell suspension is then added slowly in the flask (or dish) already containing cell culture medium.
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Waste Disposal:• Anaspirationsetupisshownbelow.Aplastictubingwitha“Vacuum-guard”filterispluggedontoalarge5l
glass vacuum flask. A common setup consists of a 5 ml plastic pipette inserted into a large rubber stopper. The addition of liquid soap on the outside of the pipette will facilitate its insertion into the hole of the rubber stopper.The pipette is then cut at its top (to remove the section with the cotton plug) and at its tip to widen the opening (this will prevent clogging). In order to snap the pipette evenly, pre-mark the plastic of the pipette with a scissor. The aspiration tubing is connected to the top portion of the pipette.
Aspirating Pipette:Aspiration pipettes are connected to this tubing during operation. The recipient flask should contain bleach so all liquid waste is disinfected as it gets into the flask. Liquid waste accumulating in this flask should be disposed of daily. MAKE SURE THIS FLASK IS PLACED IN A SECONDARY PLASTIC CONTAINER IN CASE OF OVERFLOW.
• Biohazardouswasteshouldbedisposedofinpropercontainersaccordingtoyourinstitutionguidelines.Below, an example is shown of the containers used in a tissue culture room. Make sure to always dispose of contaminated glass in hard plastic biohazard containers.
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ONE LAST IMPORTANT RECOMMENDATION WHILE CULTURING CELLS: AVOID DISTRACTIONS.
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References
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Cell Line Protocol
Acknowledgements:The NCI PS-OC program staff would like to thank Drs. Thea Tlsty and Philippe Gascard (Princeton PS-OC; UCSF) for significant contributions to this protocol book. Valuable input was also obtained from Mrs. Chira Chen-Tanyolac and Drs. Luis Estevez-Salmeron and Steve Oh (Princeton PS-OC; UCSF). The NCI PS-OC program staff would also like to thank the PS-OC Network for participating in this ongoing exercise.
