Cancer Cell Line Cultures in Xeno-Free and Serum-Free Conditions

Conventionally, established cell lines have been cultured almost exclusively in standard culture media, supplemented with substantial amounts (2%–20%) of fetal calf serum (FCS). Similar to other undefined media supplements, fetal calf serum has undesirable genetic, physiological, and epigenetic cellular effects [1–4] and has been proven to cause substantial experimental variability and misrepresentative results; for example, in hormone-related studies and drug screenings [5].

To attain a universal, stable, and xeno-free environment for culturing human cancer cell lines, the PromoCell Cancer Cell Line Medium XF was engineered as a defined formulation. With the exception of highly purified human plasma-derived albumin, it has no ill-defined components (such as extracts, fetal calf serum, or hydrolysates) and displays very low variability from one lot to another. As the formulation does not include any cellular attachment factors, it is essential to coat culture vessels with vitronectin, fibronectin, or other appropriate attachment substrates. Being widely usable across adherently growing cancer cell lines, the new culture medium is an economical solution for ensuring efficient, truly standardized routine cultures.

Expansion performance of the HT1080 fibrosarcoma cell line in the Cancer Cell Line MediUm XF compared with conventional culture conditions. HT1080 cells were plated with 5000 cells/cm2 in Cancer Cell Line Medium XF on fibronectin-coated vessels (red) or in DMEM + 2 mM L-Glutamine + 10% FCS (grey). Subsequently, the cells were cultured for 10 consecutive passages with a passage interval of 3–4 days. B, C: Morphology of HT1080 fibrosarcoma cells cultured in Cancer Cell Line Medium XF. Exemplary images of HT1080 on day 3 after subculture (P7) are shown in the Cancer Cell Line Medium XF (B) and conventional culture conditions (C) (100x magnification).

Expansion performance of the HT1080 fibrosarcoma cell line in the Cancer Cell Line MediUm XF compared with conventional culture conditions. HT1080 cells were plated with 5000 cells/cm2 in Cancer Cell Line Medium XF on fibronectin-coated vessels (red) or in DMEM + 2 mM L-Glutamine + 10% FCS (grey). Subsequently, the cells were cultured for 10 consecutive passages with a passage interval of 3–4 days. B, C: Morphology of HT1080 fibrosarcoma cells cultured in Cancer Cell Line Medium XF. Exemplary images of HT1080 on day 3 after subculture (P7) are shown in the Cancer Cell Line Medium XF (B) and conventional culture conditions (C) (100x magnification).

Figure 1A. Expansion performance of the HT1080 fibrosarcoma cell line in the Cancer Cell Line Medium XF, compared with conventional culture conditions. HT1080 cells were plated with 5000 cells/cm2 in Cancer Cell Line Medium XF on fibronectin-coated vessels (red) or in DMEM + 2 mM L-Glutamine + 10% FCS (gray). The cells were cultured for ten consecutive passages with a passage interval of 3–4 days. B, C: The morphology of HT1080 fibrosarcoma cells cultured in Cancer Cell Line Medium XF. Exemplary images of HT1080 on day three after subculture (P7) are shown in the Cancer Cell Line Medium XF (B) and conventional culture conditions (C) (100x magnification).

Background

For budgeting reasons, everlasting cancer cell lines are extensively chosen over primary cells in research. They are established, distinct model systems that act as a continuous source of cells, while avoiding the confines posed by the finite lifetime of standard primary cells [6–9]. As long as the restrictions inherent in substituting cell lines for primary cells are taken into consideration, tumor cells can be employed in in vitro models to reflect some aspects and functionalities of terminally differentiated primary human cells [7].

However, weakly defined culture media components, such as fetal calf serum, are a recognized source of result variations [1–5]. This can cancel out the benefits of using cancer cell lines as a source of cells, while also jeopardizing the reliability of experimental results.

It has been established for hormone-responsive cell lines, immune-system-related cell types and stem cells, that unclear conventional culture media can obstruct cell properties and experimental readouts. It can occur through the misrepresentation of their responses to drugs, through triggering unspecific false immune responses or through causing undesirable differentiation of stem cells [1, 3, 10]. Cells can also be considerably altered by culture conditions of this type [11].

A controlled culture environment is therefore paramount for attaining more accurate results with cell line models, and this kind of environment also allows more accurate data analysis and interpretation [4], thus taking a step forward in manipulating the unique features of permanent cell lines as economical in vitro research models.

Supplementary Data

Morphology of MCF-7 breast carcinoma cells in the Cancer Cell Line Medium XF. MCF-7 cells were plated at 10,000 cells per cm2 in fibronectin-coated vessels and cultured for three passages in the Cancer Cell Line Medium XF. The cells exhibited efficient proliferation as well as a typical—but slightly more compact—morphology as compared to traditional culture media (not shown). The image was taken five days after seeding at 100x magnification.

Figure 2. Morphology of MCF-7 breast carcinoma cells in the Cancer Cell Line Medium XF. MCF-7 cells were plated at 10,000 cells per cm2 in fibronectin-coated vessels and cultured for three passages in the Cancer Cell Line Medium XF. The cells exhibited efficient proliferation as well as a typical—but slightly more compact—morphology as compared to traditional culture media (not shown). The image was taken five days after seeding at 100x magnification.

Use Aseptic Techniques and a Laminar Flow Bench

(A) Switching an Existing Cell Culture to the Cancer Cell Line Medium XF

This protocol illustrates how a human cancer cell line in an existing culture can be converted to the Cancer Cell Line Medium XF for the first time.

I. Materials

  • Cancer Cell Line Medium XF (C-28077)
  • Proliferating culture of a human cancer cell line in good condition
  • Adhesion factors: Human Fibronectin (C-43060) or vitronectin (C-69201)
  • Phosphate buffered saline (PBS) w/o Ca2+/Mg2+ (C-40232)
  • Tissue-culture-treated cell culture vessels
  • Accutase (C-41310) or, optionally, DetachKit (C-41210)

II. Culture Protocol

1. Coat the culture vessel

The serum- and xeno-free formulation does not have any attachment factors. Therefore, it is customarily essential to coat the surface of the cell culture vessel with the right adhesion factor. Table 1 illustrates an overview of cell lines and surface coatings tested using the Cancer Cell Line Medium XF.

Table 1. Overview of cell lines and surface coatings tested with the Cancer Cell Line Medium XF. Cells were seeded at a density of 10,000 cells/cm2.

Tissue Tested Cell Line Cell Line Origin Remarks
Brain BV2 immortalized murine primary microglial cells Coat with Fibronectin (C-43060):
1 μg/cm²
Breast MCF-7 pleural effusion of metastatic human breast adenocarcinoma Coat with Fibronectin (C-43060):
1μg/cm²
Colon HT-29 human colon adenocarcinoma Coat with Vitronectin (C-69201):
0.5μg/cm²
Connective tissue HT 1080 human fibrosarcoma Coat with Fibronectin (C-43060):
1μg/cm²
Liver HepG2 hepatocellular carcinoma of the human liver Coat with Vitronectin (C-69201):
0.5μg/cm²
Lung A-549 human lung carcinoma Coat with Vitronectin (C-69201):
0.5 μg/cm²
Prostate LNCaP lymph node metastasis of human prostate adenocarcinoma 3D culture in C-28070 is recommended

To establish the culture settings, it is suggested that fibronectin and vitronectin coatings are tested. The culture vessel should be coated with 10 μg/mL human (or bovine) fibronectin or 5 μg/mL vitronectin in line with the instruction manual of the product. 100μL of diluted coating solution per cm2 of culture surface should be used. (Final concentration: fibronectin 1µg/cm2 and vitronectin 0.5µg/cm2)

Note: If not using straightaway, it is possible to store the sealed vessel for up to three months at 2–8 °C.

2. Harvest cells from the current culture

The cells should be harvested and counted from a proven culture of the right cell line using a standard technique. They then have to be re-suspended in Cancer Cell Line Medium XF.

3. Plate the cells

The cells need to be plated at the appropriate density (5000−10,000 cells/cm2). While seeding the cells in the Cancer Cell Line Medium XF for the first time, approximately 200µL of medium per cm2 of culture surface should also be used; for example, 5mL for a T25 flask.

4. Allow the cells to grow

The plated cells have to be incubated at 37 °C and 5% CO2. The medium has to be changed every 2−3 days.

Note: Cell cultures do not need to be adapted to the Cancer Cell Line Medium XF. With certain cell lines, proliferation may be slightly lower after starting the culture, but this should regularize after one to three passages.

5. Cell subculture

After the cells have reached 70%−80% confluence, the culture has to be washed two times with ambient tempered PBS w/o Ca2+/Mg2+. The cells then should be incubated for 5–10 minutes with 150µL/cm2 Accutase at 37 °C. After the initial 5 minutes of incubation, the detachment process should be monitored visually.

When the cells begin to detach, their complete dislodgement must be facilitated by tapping the flask. The same volume of Cancer Cell Line Medium XF should be added to the detached cells and spun down for 5 minutes at 300 x g at RT. The supernatant has to be carefully aspirated, and the cell pellet needs to be gently re-suspended in a sufficient amount of Cancer Cell Line Medium XF. The cells then should be seeded into new fibronectin-coated vessels and incubated further at 37 °C and 5% CO2.

Approximately 300–400µL of medium per cm2 of culture surface should be used for the following cultivation. Incubation of the cultures needs to be continued at 37 °C and 5% CO2.

(B) Routine Culture Using the Cancer Cell Line Medium XF

This protocol illustrates the standard culture of a human cancer cell line already transferred to the Cancer Cell Line Medium XF.

I. Materials

  • Cancer Cell Line Medium XF (C-28077)
  • Proliferating culture of a human cancer cell line in good condition
  • Adhesion factors: Human Fibronectin (C-43060) or vitronectin (C-69201)
  • Phosphate buffered saline (PBS) w/o Ca2+/Mg2+ (C-40232)
  • Tissue-culture-treated cell culture vessels
  • Accutase (C-41310) or, optionally, DetachKit (C-41210)

II. Culture Protocol

1. Coat the culture vessel

The culture vessel needs to be coated with the appropriate adhesion factors, as previously tested.

2a. Harvest cells from the current culture

The cells should be harvested and counted from a proven culture of the right cell line using a standard technique. They then need to be re-suspended in Cancer Cell Line Medium XF.

2b. Thaw cells from cryo-stock

Cryopreserved cells cultured beforehand in Cancer Cell Line Medium XF may also be thawed using this medium.

3. Plate the cells

The cells need to be plated at the appropriate density (5000−10,000 cells/cm2). Approximately 300−400µL of Cancer Cell Line Medium XF per cm2 of culture surface can be utilized, for example, 7.5−10mL for a T25 flask.

4.  Allow the cells to grow

The plated cells have to be incubated at 37 °C and 5% CO2. The medium has to be changed every 2–3 days.

5. Cell subculture

After the cells have touched 70%–80% confluence, the culture has to be washed twice with ambient tempered PBS w/o Ca2+/Mg2+, and the cells have to be incubated for 5–10 minutes with 150 µL/cm2 Accutase at 37 °C. After the initial 5 minutes of incubation, the detachment process should be tracked visually.

When the cells begin to detach, their complete dislodgement should be facilitated by tapping the flask. The same volume of Cancer Cell Line Medium XF should be incorporated into the detached cells and spun down for 5 minutes at 300 x g at RT. The supernatant has to be carefully aspirated and the cell pellet gently re-suspended in a sufficient amount of Cancer Cell Line Medium XF. The cells have to be seeded into new coated vessels and further incubated at 37 °C and 5% CO2. Approximately 300–400µL of medium per cm2 of culture surface must be used. The incubation of the cultures must be continued at 37 °C and 5% CO2.

Products

Product Size Catalog Number
Cancer Cell Line Medium XF 250ml C-28077
Fibronectin Solution, human (1 mg/ml) 5ml C-43060
Vitronectin, human, recombinant (HEK) 500μg C-69201
Accutase-Solution 100ml C-41310

Related Products

Product Size Catalog Number
Dulbecco’s PBS, w/o Ca++/Mg++ 500ml C-40232
Fibronectin Solution, bovine (1 mg/ml) 5ml C-43050
DetachKit 3 x 125ml C-41210
Cryo-SFM 30ml / 125ml C-29910 / C-29912

References

[1] Sulit, H.L., et al., Human tumor cells grown in fetal calf serum and human serum: influences on the tests for lymphocyte cytotoxicity, serum blocking and serum arming effects. Int J Cancer, 1976. 17(4): pp. 461-8.

[2] Darro, F., et al., Characterization of the differentiation of human colorectal cancer cell lines by means of Voronoi diagrams. Cytometry, 1993. 14(7): pp. 783-92.

[3] Sedelaar, J.P.M. and J.T. Isaacs, Tissue Culture Media Supplemented with 10% Fetal Calf Serum Contains a Castrate level of Testosterone. The Prostate, 2009. 69(16): pp. 1724-1729.

[4] Baker, M., Reproducibility: Respect your cells! Nature, 2016. 537(7620): pp. 433-435.

[5] Sikora, M.J., et al., Endocrine Response Phenotypes Are Altered by Charcoal-Stripped Serum Variability. Endocrinology, 2016. 157(10): pp. 3760-3766.

[6] Kim, H.S., Y.J. Sung, and S. Paik, Cancer Cell Line Panels Empower Genomics-Based Discovery of Precision Cancer Medicine. Yonsei Med J, 2015. 56(5): pp. 1186-98.

[7] Masters, J.R., Human cancer cell lines: fact and fantasy. Nat Rev Mol Cell Biol, 2000. 1(3): pp. 233-6.

[8] Kondo, T., Stem cell-like cancer cells in cancer cell lines. Inflammation and Regeneration, 2007. 27(5): pp. 506-511.

[9] Fillmore, C.M. and C. Kuperwasser, Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res, 2008. 10(2): pp. R25.

[10] Hurt, E.M., et al., Identification of Vitronectin as an Extrinsic Inducer of Cancer Stem Cell Differentiation and Tumor Formation. Stem Cells (Dayton, Ohio), 2010. 28(3): pp. 390-398.

[11] Martin, M.J., et al., Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med, 2005. 11(2): pp. 228-32.

About PromoCell

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With more than 28 years of experience in human primary cell culture, we deliver research tools for scientists worldwide. Because sharing knowledge is key to scientific discovery, we also offer professional training at our PromoCell Academy. Our team of experts is by your side in your quest for new therapies.


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Last updated: May 21, 2019 at 10:15 AM

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