Primary Cell Culture Support—Getting Started

When cells are isolated from a tissue to form a primary culture, assuming that the cells proliferate in vitro, a confluent monolayer or a dense cell suspension is formed. According to the traditional definition, the first harvesting and subculture of this cell population results in the formation of a cell line [Freshney, R.I. (1987). Culture of Animal Cells. A Manual of Basic Technique. (New York, Alan R. Liss, Inc.)]. This type of cell line has a finite lifespan, during which cells with the highest growth capacity will predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. 

Using this nomenclature system, a continuous cell line is a population of cells that has undergone a genetic transformation, resulting in indefinite growth potential. Continuous cell lines are usually aneuploid. In practice, continuous cell lines can be cultured through a very high number of subcultures, although some further genotypic, and therefore phenotypic, changes may occur at very high passage numbers. Immortalization can occur spontaneously or may be virally- or chemically- induced. Keep in mind that the working definitions of these terms can vary between research groups. Many researchers do not use the term “cell line” to refer to any population unless it has undergone a genetic transformation. 

A cell strain is a subpopulation of a cell line that has been positively selected from the culture, by cloning or some other method. A cell strain will often have undergone additional genetic changes since the initiation of the parent line. Individual cell strains may, for example, have become more or less tumorigenic than the established line, or they may be designated as a separate strain following transfection procedures. 

The term cell type refers to all cells with a common phenotype, e.g., keratinocyte, melanocyte. Therefore keratinocytes isolated from a number of different donors are all the same cell type. 

"Normal" means that the cells in question were isolated into primary culture from normal healthy tissue, rather than from diseased tissue. "Normal" also refers to a cell population that constitutes a cell line as opposed to a continuous cell line according to the traditional definitions given above, since the cells have not been genetically altered and do not have indefinite growth potential. 

A population doubling is a two-fold increase in the total number of cells in a culture, and is most commonly referred to during the exponential, or “log”, phase of growth. 

The term passage number refers to the number of times that a cell population has been removed from the culture vessel and undergone a subculture (passage) process, in order to keep the cells at a sufficiently low density to stimulate further growth. ForInvitrogen™ cultured cells, the first culture following the isolation of cells from tissue is termed the primary culture. After the first subculture, the cells are described as a secondary culture (or passage 1). After the second subculture, the cells become a tertiary culture (or passage 2), and so on. 

See our detailed protocol for counting cells using a hemacytometer.

 The procedure given below is a sample protocol for establishing cultures from the contents of one vial. 

1. Prepare a beaker of water at 37°C. 
2. Remove a vial of cells from liquid nitrogen storage, taking care to protect hands and eyes. 
3. Loosen the cap on the vial 1/4 turn for 10 seconds to release any liquid nitrogen that may be trapped in the threads, then re-tighten the cap. 
4. Dip the lower half of the vial into the 37°C water to thaw. 
5. When the contents of the vial have thawed, wipe the outside of the vial with disinfecting solution and move to a Class II, type A laminar flow culture hood. 
6. Open the vial and pipette the suspension up and down with a 1 mL pipette to disperse the cells. 
7. Remove 20 µL from the vial and dilute the cell suspension in 20 µL of trypan blue solution (for example: Gibco™ Trypan Blue, Cat. No. 15250-061). 
8. Use a hemacytometer to determine the number of viable cells per mL. 
9. Dilute the contents of the vial (1 mL) to the concentration recommended by the product instructions (for example 1.25 x 10E4 viable cells/mL for Gibco™ neonatal human epidermal keratinocytes ). 
10. Add 5 mL of cell suspension to each 25 cm² culture flask or 15 mL of cell suspension to each 75 cm² culture flask. 
11. Following inoculation, swirl the medium in the flasks to distribute the cells. Many cell types attach to culture surfaces quickly, and if the medium is not distributed immediately following inoculation, the cells may grow in uneven patterns. 
12. Incubate the cultures in a 37°C, 5% CO²/95% air, humidified cell culture incubator. For best results, do not disturb the culture for at least 24 hours after the culture has been initiated. 

We do not recommend spinning cells out of cryopreservation medium prior to plating. Centrifugation can be harmful to cells, particularly if inappropriately high speeds are used. Experience in our Invitrogen™ cell culture laboratory has shown that cells do not suffer deleterious effects if the DMSO concentration is sufficiently low. Therefore, our product instructions include a detailed protocol that involves diluting the cells into culture medium such that the final DMSO concentration is less than 0.4% (v/v) at the recommended seeding density and volume of medium. 

When either Gibco™ or Invitrogen™ cryopreserved or proliferating cultures are purchased from Thermo Fisher Scientific, they may be expanded and cryopreserved again. However, the cryopreservation process may result in altered growth performance of the cells. The following protocol provides a basic guideline for the cryopreservation of cells using Synth-a-Freeze™ medium, a defined, protein-free cryopreservation medium available from Thermo Fisher Scientific. 

Please note: Due to differences in cryopreservation equipment and individual techniques, we cannot guarantee that cells cryopreserved using this protocol will be viable upon recovery from cryopreservation, and we do not provide a warranty for cells cryopreserved in an investigator's laboratory. 

1. Thaw Synth-a-Freeze™ medium in a 37°C water bath or overnight at 4°C. 
2. If thawed in a water bath, do not exceed 37°C and do not leave the product at 37°C for an extended period of time. 
3. Synth-a-Freeze™ medium should be equilibrated to 4°C prior to use. For optimal results, the use of a controlled-rate freezer is recommended. In the absence of a controlled-rate freezer, a cell cryopreservation container (e.g., Thermo Scientific™ Mr Frosty™ container) may be useful. 
4. If enzymatic agents are used to remove the cells from a culture surface, resuspend the cells in a solution that will neutralize the effects of the enzyme. 
5. Pellet the cells by centrifugation. 
6. After removing the supernatant, resuspend the cell pellet in cold Synth-a-Freeze™ medium at a concentration of 5 x 10E5 to 3 x 10E6 cells/mL. 
7. Distribute the cell suspension in an appropriate number of cryopreservation vials. 
8. Cool the vials of cells to 4°C as quickly as possible. 
9. If using a controlled-rate freezer: freeze the material by reducing the temperature 1°C per minute until the temperature reaches –40°C. Then reduce the temperature at a rate of 2°C per minute until the temperature reaches approximately –90°C. 
10. If using a cell cryopreservation container: Prepare the container according to the manufacturer’s instructions. 

For best results we recommend transferring the vials to the vapor phase of a liquid nitrogen storage facility as soon as possible after the cells have reached –80°C. 

As a substitute for Synth-a-Freeze™ medium, the recommended basal medium for the cell type being cryopreserved, supplemented with 10% fetal bovine serum (FBS) and 10% DMSO, may be used. Please note that Synth-a-Freeze™ medium is NOT recommended for the cryopreservation of human epidermal melanocytes. 

Cells should be pelleted by centrifugation at 180 x g (relative centrifugal force, RCF). The correct rotor speed can be calculated by measuring the maximum radius of your rotor and entering the information into the table found at this website. For example, to centrifuge cells at 180 x g (RCF) in a rotor with a radius of 15 cm, you would spin the cells at 1,036 rpm. 

Yes, our primary cells are isolated from tissue using enzymatic digestion. 

No, the tissue was not cryopreserved.

It varies a lot depending on the age of donor, size of tissues, locations, and conditions arrived to our facility. Sometime a small amount of tissue gives us many cells and sometimes a large amount of tissue gives us a very small lot. Typically, we can get 30–300 vials. 

Cumulative population doublings are calculated from the seeding density and harvesting density from each culture level. 

We use Medium 106 and LSGS.

The image below are of HCECs imaged using the Click-iT™ Edu Alexa Fluor™ 488 imaging kit, anti-alpha-tubulin antibody with goatd anti-mouse Alexa Fluor™ 555 secondary and Hoechst™ 3342 dye.

We recommend culturing HCECs in keratinocyte serum-free medium (KSFM), as the cells are manufactured and quality-controlled using this medium. However, if a defined growth system is preferred, we recommend defined keratinocyte serum-free medium (Defined Keratinocyte-SFM). Note that a Coating Matrix Kit is required for culturing 

HCECs in Defined Keratinocyte-SFM. In-house testing has shown comparable HCEC growth and morphology in both KSFM and Defined Keratinocyte-SFM.

Feeder cells are not needed for culturing HCECs using KSFM or Defined Keratinocyte-SFM. However, HCECs cultured in Defined Keratinocyte-SFM require the use of a Coating Matrix Kit (recombinant type I collagen) for efficient attachment and growth.

When cultured under recommended conditions, HCECs grown in KSFM typically achieve growth rates of ~0.75–1.0 population doublings per day. Similar growth rates are seen using Defined Keratinocyte-SFM.

HCECs seeded at 5,000 cells/cm² typically reach ~90% confluency in 5–7 days when cultured as recommended.

Each lot of HCECs is tested for positive expression of the widely accepted corneal epithelial markers p63α and cytokeratin 15 (CK15).

HCECs are isolated from normal human corneal-scleral tissue. Trimmed limbal regions are enzymatically digested, and released epithelial cells are expanded. HCECs are cryopreserved using a controlled-rate freezer at the end of the secondary culture (p1) in a cryopreservation reagent containing 10% dimethyl sulfoxide (DMSO).

HCECs are performance-tested and guaranteed to reach ≥12 population doublings after thaw. Generally, this requires 3–4 passages when HCECs are seeded at 5,000 cells/cm².

In-house evaluation of BacMam (recombinant baculovirus-encoding mammalian expression cassette) delivery technology has shown highly efficient expression in HCECs. BacMam 2.0 reagents, including CellLight™ fluorescent protein–signal peptide fusions and other BacMam-based biosensor products typically yield transduction efficiencies of >70% under optimized conditions.

HCECs display the typical cobblestone morphology observed with many types of epithelial cells grown in monolayer culture. Refer to the image on the HCEC product sheet for a representative phase-contrast image of cells approaching ~90% confluence. When cells are continually passaged, it is normal to see changes in morphology, including noticeable increases in average cell size. Cells may also appear more flattened and skirted. A reduction in the mitotic index or growth rate is usually observed in higher-passage cultures.

Please see the image below of phase contrast images of neonatal Human Dermal Fibroblasts (HDFn) in culture:

Day 3 - HDFn

Day 5 - HDFn

Day 7 - HDFn

HDFn cells are grown in Medium 106 with the addition of Low Serum Growth Supplement. 

Please see the image below of cells after 48 hours that were thawed and plated in DMEM plus 2% hose serum:

Please see the images below showing Gibco™ Human Skeletal Myoblasts responding to physiological levels of TGF-β1 and IGF-1. TGF-β1 addition inhibits differentiation, whereas the addition of IGF-1 (Cat. No. PHG0075) blunts this effect. The anti-troponin staining method was used.

These cells come from human skin:

They are both the same. Keratinocytes are referred to as basal cells or basal keratinocytes.

This information also appears on the COA.

We recommend coating the plates with this kit for all HEK grown in Animal Product–Free (APF) products.

Each lot is tested for doubling time and the results are listed on the COA.

These are senescent cells, and this is normal for adult keratinocyte culture. You always have a population of cells that are old and no longer proliferate. However younger cells, which are small in size, should keep proliferating. When a culture gets older, you see more and more large cells, and the culture will eventually stop growing. With the right care and attention, the culture should yield at least 25 population doublings. 

The picture below shows a confluent monolayer of keratinocytes:

See the image below of our human umbilical vein entothelial cells grown in Medium 200 with the addition of Low Serum Growth Supplement (LSGS).

Day 3 - HUVEC

Day 5 - HUVEC

Day 7 - HUVEC

We carry primary human umbilical vein endothelial cells (HUVEC) from one donor or pooled from multiple donors.

The image below is of our human mammary epithelial cells (HMEC) grown in HuMEC medium with the addition of HuMEC growth supplement.

Day 1 - HMEC

Day 3 - HMEC

Day 5 - HMEC

Yes. HMEC are isolated from normal, human reduction mammoplasty tissue.

HMEC are guaranteed for a minimum of 16 population doublings after thaw.

HMEC usually have population doubling times of 24–30 hr during the guaranteed lifespan.

One passage is equivalent to ~4 population doublings when using the recommended subculture conditions.

Procedures for 3D culture, BacMam gene delivery, transfection, as well as with immunocytochemical staining (ICC), can be found here. 

Both media are designed for culture of HMEC. However, in-house studies show HuMEC Ready Medium (Thermo Fisher Scientific Cat. No. 12752-010) outperforms M171/MEGS (Thermo Fisher Scientific Cat. No. M-171-500/S-015-5) in both growth rate and lifespan measurements of HMEC. There is no difference between media when staining for cell-specific markers using indirect immunocytochemical staining ICC.

One vial (500,000 viable cells) can seed eight T-25 flasks at 2,500 viable cells per cm². A hemocytometer should be used to accurately count cells and to help ensure an appropriate seeding density. 

No. HMEC cultures are easily initiated at 2.5 x 10E3 cells per cm² in standard tissue culture plasticware and passaged using a trypsin/EDTA solution.

The image below shows HEMa cells grown in Medium 253 with the addition of Human Melanocyte Growth Supplement (first culture after thaw):

Day 5 - HEMa

Day 9 - HEMa

Day 14 - HEMa

Do not use Synth-a-Freeze™ Cryopreservation Medium to cryopreserve melanocytes. We recommend using DMEM with 10% FBS and 10% DMSO, or the Recovery Cell Culture Freezing Medium.

Please see the image below of HMVECad cells grown in Medium 131 with attachment factor with the addition of Microvascular Growth Supplement (first culture after thaw):

Day 3 - HMVECad

Day 5 - HMVECad

Day 7 - HMVECad

Primary rat cortex and hippocampus neurons were isolated from cortex or hippocampus tissues (respectively) of 18-day embryos (E-18) from Fisher 344 rats and directly cryopreserved after isolation.

Neurite extensions will be seen beyond 4 days post thaw. The longer the cells are in culture, the longer the neurite extensions.

These cells can be cultured for months. However, they do not proliferate and will not revive if they are cryopreserved.

The exposure of neurons to air will result in cell death and low overall culture viability.

Gibco™ Human Astrocytes were isolated from fetal brain (17–24 weeks of gestation) and cryopreserved at passage 1. It is tissue-specific. Donor information (if available) is stated on the Certificate of Analysis.

Human astrocytes can proliferate for a limited amount of time. The culture could have 3–15 fold increase in cell number over 10 days in the recommended medium. However, we do not recommend cryopreserving the cells after initial thaw.

No. These cells must be grown in Geltrex™ Matrix–coated tissue culture vessels.

Rat cortical astrocytes were isolated from the cortex of fetal (E-19) Sprague-Dawley rats. The cells were cryopreserved in growth medium supplemented with 10% DMSO at passage 1.

Rat cortical astrocytes can be expanded in culture for at least one passage. The recommended medium can provide 3–15 fold increase in cell number over 10 days. For consistent results in your experiments, we recommend using cells below passage 3 (P3). If you expand these cells beyond P3, we recommend that you perform another round of characterization prior to further experiments.

These cells readily stick to the plastic used in cell culture dishes and centrifuge tubes. Prior to use, rinse all materials that will come in contact with the cells with medium to prevent cells from sticking to the plastic.

Rat Glial Precursor Cells were isolated from the cortex of newborn Sprague Dawley rats at day 2 after birth, and cryopreserved at passage 2. 

These cells can be thawed and passaged once before using in experiments. The single passage will yield a 2-fold increase of thawed cells. Cells do not expand significantly beyond first passage post-thaw.

Neurobasal Medium is optimized for prenatal and fetal neurons. Neurobasal-A Medium is optimized for growing postnatal and adult brain neurons. These two media differ only in osmolality. 

• As a general guideline, we recommend using Gibco™ B-27™ Supplement to culture neural stem cells, hippocampal, and other CNS neurons.
• For neuroblastomas, or post-mitotic neurons from both PNS and CNS, Gibco™ N-2 Supplement can be used. 
• For primary glial cells (astrocytes) or tumor cell lines of astrocytic phenotype (astrocytes and gliomas), or oligodendrocytes, Gibco™ G-5 Supplement can be used.

The yellow color comes from vitamin components.

We offer Gibco™ Hibernate™ Medium for this purpose. When supplemented with Gibco™ B-27™ Supplement and Gibco™ GlutaMAX™-1 Supplement, this medium can allow for the manipulation of neurons at ambient CO₂ for at least 48 hours while retaining their viability, and preserve viable brain tissue for up to a month when stored at 4°C. Two Hibernate™ media are offered:

• Gibco™ Hibernate™-A Medium (Cat. No. A1247501) is formulated for postnatal neurons.
• Gibco™ Hibernate™-E Medium (Cat. No. A1247601) is formulated for embryonic neurons.

These two media have the same formulation and differ only in osmolality.

L-Glutamate is excitotoxic as neuron cells mature. For primary hippocampal neurons and other embryonic neurons, we recommend that you add 25 µM L-Glutamate to the initial plating medium. However, after day 4, L-Glutamate should not be added, as it is toxic to neuron cells beyond day 4.

For neuroblastomas, L-Glutamate should be included in the medium for both plating and subsequent medium changes.

We offer the following B-27 supplement categories, which are developed for specific applications:

Gibco™ B-27™ Serum-Free Supplement (Cat. No. 17504044): Complete formulation for the growth of long-term viability of neurons.

• Gibco™ B-27™ Supplement minus AO (antioxidants) (Cat. No. 10889038): Five antioxidants are removed. The supplement is ideal for studies of oxidative damage, apoptosis, or applications in which free radical damage to neuron occurs.
• Gibco™ B-27™ Supplement minus Insulin (Cat. No. A1895601): Insulin is removed. The supplement is ideal for studies of insulin secretion and insulin receptors.
• Gibco™ B-27™ Supplement minus Vitamin A (Cat. No. 12587010): Vitamin A—which induces neural differentiation—is removed. The supplement is ideal for proliferation of stem cells.

We also offer Gibco™ B-27™ Electrophysiology Kit (Cat. No. A1413701) for electrophysiology experiments.

The B-27 Electrophysiology Kit is optimized for electrophysiology experiments, and developed to enhance network spike rates. It contains extra components which help to increase spike rate and enhance synaptogenesis.

No, primary rat neurons do not proliferate.

Primary rat neurons cannot be cryopreserved again because they will not revive.

Glutamate helps hippocampal neurons sprout and promotes viability. However, L-glutamate can only be supplemented in the medium up to day 4 in culture because it is toxic to mature neurons.

Please view the images below of HASMC grown in Medium 231 and Smooth Muscle Growth Supplement:

Day 5 - HASMC

Day 7 - HAMSC

Day 9 - HAMSC

This is a blood glycoprotein involved in hemostasis. It is constitutively produced in endothelium, megakaryocytes, and subendothelial connective tissue and constantly present in the blood plasma. Von Willebrand factor plays a major role in blood coagulation. 

Smooth muscle cells should be negative for VWF and positive for a-actin. Testing for this component is an indication of the purity of the culture.

Angiogenesis—the formation of new blood vessels from existing vasculature—is an integral part of both normal and pathological processes. It is required for tumor growth and metastatic spread, and as a result is a hot research area within oncology. This complicated process results from the input of multiple signaling pathways. 

During angiogenesis, endothelial cells disrupt the surrounding basement membrane, migrate toward an angiogenic stimulus, proliferate to form the new vessel, and reorganize to create the necessary three dimensional vessel structure. In vitro assays are widely used to study these functions in the presence of either angiogenic or antiangiogenic agents. 

Cryopreserved hepatocytes are shipped in the vapor phase of liquid nitrogen contained in a non-hazardous container called a dry liquid nitrogen (LN2) vapor shipper or dewar. The internal temperature of the dewar is maintained between –140°C and –160°C, generally for up to 7–10 days if unopened. Once hepatocytes are received and transferred to a long term LN2 dewar in the laboratory, dewers are returned to the address listed on the pre-paid return shipping label for reuse. 

Upon receipt of a shipment of cryopreserved hepatocytes, carefully and quickly transfer the vials to the vapor phase of liquid nitrogen and keep at –135°C or below until use. Any increase in the temperature of the cryovials before an experiment threatens the viability, functionality, and activity of the hepatocytes. 

For additional information, please refer to the ADME/Tox Support Center.

If properly stored, cryopreserved hepatocytes can be maintained in the vapor phase of liquid nitrogen (–135°C or below) for several years. This makes them ideal for a series of experiments conducted over many months.

For additional information, please refer to the ADME/Tox Support Center.

Unlike immortalized cell lines, hepatocytes are primary cells that cannot be cultured indefinitely. The use of thawed suspension hepatocytes should be limited to short-term experiments with a maximum of 4–6 hour incubations. Plateable hepatocytes, which attach to collagen-coated plasticware in culture media, are generally metabolically active for anywhere from 2–7 days depending on which assay they are qualified for use.

For additional information, please refer to the ADME/Tox Support Center.

No. The overlay will interfere with transfection. For transfection assays, we recommend that you perform the transfection first, and then proceed to overlay the cells.

We recommend putting the Gibco™ HepExtend™ Supplement through no more than two freeze/thaw cycles. Therefore, we recommend that Gibco™ HepExtend™ Supplement be thawed and divided into single-use aliquots, which are then stored in a non–frost-free freezer at –20°C to –5°C.

For additional information, please refer to the ADME/Tox Support Center.

Gibco™ HepExtend™ Supplement does not contain growth factors, cytokines, FBS, or pharmaceutical small molecules. However, it does contain small amounts of BSA (0.12375g/mL at 50X).

For additional information, please refer to the ADME/Tox Support Center.

Gibco™ HepExtend™ Supplement is compatible with current hepatocyte culturing methods and instrumentation and therefore, we recommend following the Gibco™ Thawing and Plating Cryopreserved Hepatocytes protocol that is available on our website.

For additional information, please refer to the ADME/Tox Support Center.

We recommend that Gibco™ HepExtend™ Supplement be used to keep hepatocytes in culture for up to 10 days, and it has been shown to work on approximately 80% of human lots tested—including metabolism-qualified, induction-qualified, and transporter-qualified lots.

For additional information, please refer to the ADME/Tox Support Center.

At this time, Gibco™ HepExtend™ Supplement has only been tested on cryopreserved human hepatocytes; we therefore cannot recommend using this supplement on other species.

For additional information, please refer to the ADME/Tox Support Center.

The Gibco™ HepExtend™ Media Kit contains the Gibco™ HepExtend™ Supplement (Cat. No. A2737501) as well as Williams E Medum, 500 mL (Cat. No. A1217601) and the Hepatocyte Maintenance Supplement Packs (Cat. No. CM4000) which are all the components needed to make the complete medium for hepatocyte culture.

For additional information, please refer to the ADME/Tox Support Center.

The cells were treated with 2.5 ng/mL of TGF-β1 for 48 hr.

Propagation of stellate cells results in further activation of the cells which significantly reduces the ability of the cells to get induced by inflammatory cytokines. Hence, we do not recommended further propagation of the cells.

We have found that culturing hepatic stellate cells in collagen I-coated wells helps to maintain the morphology of the stellate cells.

We do not offer quiescent human stellate cells (q-HSCs). However, we do offer Activated Human Myofibroblastic Hepatic Stellate Cells (MF-HSCs) (Cat. No. HMFHSC).

The qHSCs only go through a few passages, just sufficient for them to get activated. However, the low number of passages maintains their ability to get induced by inflammatory cytokines.