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Basic medium:
DMEM high glucose / F-12
Or renal epithelial cell basal medium such as ATCC®PCS-400-030 Recombinant human insulin 0.5 ug/ml Required Cortisol 0.1 ug/ml Required Adrenaline 0.5 ug/ml Required Recombinant human EGF 50 ng/ml Ideal/beneficial triiodo-L-thymidine 10 pg/ml Required Recombinant human EGF 10 ng/ml Ideal/beneficial Primary hepatocyte
Basic medium:
Williams's Medium E Recombinant human insulin 5 ug/ml Required Cortisol 0.5 ug/ml Required Recombinant human EGF 50 ng/ml Ideal/beneficial Primary keratinocyte
DMEM/F-12
1:3 ratio Bovine pituitary extract (BPE) 4 ul/ml Required Cortisol 5 ug/ml Required Adrenaline 0.5 ug/ml Required Recombinant human EGF 0.125 ng/ml Ideal/beneficial Primary cardiomyocyte
Claycomb Medium T3 (triiodo-L-thymidine) 1ng/ml (1.5nM) Required Recombinant human insulin 5 ug/ml Required Recombinant human EGF 5 ng/ml Ideal/beneficial Recombinant human bEGF 5 ng/ml Ideal/beneficial Neurons
DMEM high glucose Recombinant human EGF 50 ng/ml Ideal/beneficial Recombinant human insulin 0.5 ug/ml Required
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Serum Substitute: XerumFree TM XF205 Medium Additive Instruction Manual
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The following translations are for reference only! For the end user, please refer to the original English manual.
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Enable cells to grow in a serum-free environment
Ensure animal-free, GMP- certified cell culture supplements
Serum-free culture of cells is challenging. The purpose of this article is to guide end users to a smooth transition to a serum-free environment and avoid all deficiencies or inappropriate efforts.
The ideal transition to a serum-free environment should go through several stages, gradually screening to allow cells to grow in a serum-free environment. However, if all the key steps are well resolved, a direct transition to a serum-free environment may also be successful.
Regardless of the method used, key points include the growth state of cell seeding, cell seeding density, subculture techniques, and biophysical properties of the cell culture system.
TNCbio's XerumFree TM serum replacement additive has been used as a culture medium, using conventional methods of culturing cells with the same serum. However, the concentration is 5 times the concentrate, so you can add 2% concentrate to the regular medium, the procedure is the same as usual.
content
Key considerations before use
1 preparation
1.1 Preparation of conventional cell culture media
1.2 Transition to a routine adjustment method in a serum-free environment
1.2.1 Direct transition
1.2.2 Subsequent adjustments
2 XF205 use
2.1 cell line
2.1.1 adherent-dependent cell line
2.1.2 adherent non-dependent cell line
2.2 stem cells
2.2.1 Preparation Steps - Coating
2.2.2 Cultivation of hESCs and hiPSCs
2.2.3 Cultivation of MSCs
2.3 primary cell culture
2.3.1 General recommendations
2.3.2 Specific recommendations for cell culture
2.3.3 Recommended system
Key considerations before using the concentrated XerumFree TM XF205 :
-- XF205 is a cell culture medium add-on that replaces serum and is not the final medium.
-- XF205 must be used with basal cell culture media (eg IMDM, DMEM-F12 or other basal media).
-- No need to filter the media after XerumFree TM XF205 or XerumFree TM .
- if necessary, to filter off the medium and then added to the culture medium XerumFree TM XF205 filter off the under sterile conditions.
-- compared to serum, XF205 is a 5-fold concentrate, so use the same amount (eg 2% XF205 instead of 10% FBS).
-- XF205 does not contain growth factors such as cytokines, hormones, etc., and therefore does not contain insulin.
1 preparation
1.1 General preparation method of serum-free cell culture medium
Gently and briefly shake the XerumFree TM bottle before use.
Add 5 times concentrated XerumFree TM to your basal medium (eg 10% FBS equivalent to 2% XF205 ).
Do not filter or filter off XerumFree TM medium was added finished off XerumFree TM (XF 205 is sterile).
If the medium needs to be filtered out, it needs to be filtered before adding XF.
Do not add antibiotics at this stage. In fact, antibiotics bind to serum plasmin like many other compounds, especially to albumin fragments. Therefore, the same concentration of antibiotics under serum-free and albumin-free conditions will show higher biological activity, and this increased activity may have a detrimental effect on cell growth.
In order to prevent antibiotic-free culture is not feasible, it is recommended to add 50mg/L gentamicin to the medium.
* Some users like or need to culture cells without insulin. If you use XerumFree TM to culture cells, you can choose to grow cells with or without insulin or other growth factors. If insulin is required to promote cell proliferation or performance studies, we recommend adding a final concentration of 1.25 mg/L recombinant insulin to the cell culture medium.
1.2 Conventional methods for transitioning to a serum-free environment
There are usually two ways to make cells transition to a serum-free environment for growth.
1.2.1 Direct transition
Directly transfer cells from serum-containing medium to serum-free medium.
1.2.2 Gradual adaptation
The cells were gradually transferred from serum-containing medium to serum-free medium according to the following procedure, and the serum-containing medium was halved in each step, so serum-free medium was added according to the following ratio:
Stage 1: 50% XerumFree -supplemented medium / 50% Serum-supplemented medium
Stage 2: 75% XerumFree -supplemented medium / 25% Serum-supplemented medium
Stage 3: 87.5% XerumFree -supplemented medium / 12.5% ​​Serum-supplemented medium
Stage 4: 93.75% XerumFree -supplemented medium / 6.25% Serum-supplemented medium
Stage 5: 96.88% XerumFree -supplemented medium / 3.12% Serum-supplemented medium
Stage 6: 98.44% XerumFree -supplemented medium / 1.56% Serum-supplemented medium
Stage 7: 100% XerumFree -supplemented medium
According to the above steps, if the cell growth is not good, return to the step, the cell growth returns to normal and continue to the following stage.
Cell cultures may be composed of different cell lines (adherent or suspension cells) or primary cell cultures. However, from a functional point of view, cell types may differentiate to different degrees or exhibit undifferentiated characteristics during stem cell preparation. In different situations, in order to ensure the greatest success of our cultured cells, we have to consider that different cell types have different requirements and require different transition methods.
For this reason, we divide the transition procedure into three parts, corresponding to the three types of cells:
2.1 – Cell line
2.2 – Stem cells
2.3 – primary cells
2.1 cell line
 The following protocol is valid for conventional (diploid cells, finite cell lines) or transformed or immortalized cell lines (unlimited cell lines).
2.1.1 adherent-dependent cell line
Key success factors:
--Ideal cell-adherent coating
-- Minimize trypsin
--Selection of antibiotic system
Experimental procedure
A) Coating the surface of the cell culture vessel with sufficient cell adhesion factor.
- Several commercial coating agent as Pronectin TM F, MapTRIX TM or other equivalent product
-- fibronectin or polylysine or other
- A small amount of FBS (as 500 ul added to a T25 flask) was incubated overnight at 37 ° C, followed by two washes with fresh medium or PBS.
-- A ready-to-use plastic container for cell attachment.
B) Dissociation of cells into monolayers
- The use of standard trypsin in serum-free conditions may not be feasible because serum contains trypsin inhibitors. Therefore, in order to avoid irreversible damage to cells, it is very important to minimize residual trypsin hydrolysis activity under serum-free conditions. For best results, a trypsin inhibitor (such as derived from soy) or a non-mammalian-derived dispersant such as AccutaseTM can be used, and AccutaseTM does not require inactivation or removal during the passage. In addition, most of the residual trypsin can be removed by washing cells. However, this method requires an additional centrifugation step, and centrifugation may be detrimental to certain cell types.
--Priority: Do not use trypsin, use AccutaseTM or DetachinTM to disperse cells as monolayer cells; these cell dispersions have been able to meet the requirements of gentle and effective dispersion of adherent cells; they will not damage cell membranes and surface epitopes And can maintain the structural and functional properties of the protein surface intact.
C) Cells were seeded at a density of 20000/cm 2 into complete medium prepared according to Point 1.
It is important to observe the state of high seeding density in the first step from the serum medium to the serum-free medium adaptation process. Cells generally secrete large amounts of factors that regulate cell attachment, growth and proliferation into the culture medium. However, these factors are not present in the fresh serum-free medium just after inoculation, so in order to induce the production of sufficient autocrine/paracrine factors in time, it is crucial to reach a critical value of cell density at the time of inoculation.
D) Incubate and maintain cells at 37 ° C until 80-90% confluence is reached.
At this stage, 75% of the medium was changed every 2-3 days. Do not discard the used medium, collect them, filter and store at 4 °C for the next step. If the cells appear to be stopped at any point, give more time for the cells to adapt to the new serum-free environment.
E) When approaching the confluence rate, separate the cells at a ratio of 1:2 or 1:3.
For the second stage XerumFree TM, the coating is not required, it is strongly recommended that the step of using the spent medium, because it contains the regulation of cell attachment, spreading, proliferation and autocrine growth factors. The cells were seeded in a medium containing 75% fresh serum-free medium + 25% used medium (collected in the previous step). Continue to replace with 75% fresh medium every 2-3 days to supply the cells and continue collecting the used medium according to step d).
F) Repeat step E) until the cells show similar growth dynamics compared to previous growth in serum-containing medium. At this time, the cell line can be considered to be fully adapted to serum-free culture, which may require 4-6 stages.
G) From this stage, antibiotics may be added to the medium. We recommend the use of the broad-spectrum antibiotic gentamicin; gentamicin has a lower toxicity compared to the standard penicillin/streptomycin solution, and the recommended concentration of gentamicin is 50 mg/L.
H) Once the cells are adapted to serum-free culture, the original distribution ratio (under culture conditions containing serum) should be used.
2.1 cell line
2.1.2 adherent independent cell line
The following protocol applies to cell lines grown in suspension. For transitional growth of adherent cells to a serum-free environment, refer to the TNC BIO's Technical Note "Adaptation of cells from monolayer to serum-free environmental suspension culture".
Key success factors:
Choice of antibiotic system
Experimental procedure
A) When the cell density reaches 3-5 x 10 6 cells/ml (depending on the cell line), the conversion to the XerumFree TM added medium is started. The cell suspension was collected, a small amount was taken for cell counting, and the entire suspension was centrifuged at 200 g for 5 minutes.
B) Complete cell counting
When C) in a medium added XerumFree TM in a cell density of 106 cells / ml, the cells were resuspended particles.
It is important to observe high cell seeding densities in the first step of the transition process. However, in the inoculation step, the fresh serum-free medium is free of these factors, so in order to induce the production of sufficient autocrine/paracrine factors in time, it is crucial to reach a critical value of cell density at the time of inoculation.
D) Incubate the cell culture at 37 ° C until the cell density reaches approximately 3-5 x 10 6 cells/ml.
E) by adding the appropriate volume of fresh medium to 1: 3 or 1: 4 ratio separately suspension culture (such as a cell suspension 25ml + 75ml of medium containing XerumFree TM, assigned to four separate flasks)
F) Repeat step E) until the culture shows growth dynamics as in the medium containing the serum. Since then, the cell line has been fully adapted and can be used in the original ratio in serum-containing medium.
G) Starting from this stage, antibiotics are added to the medium.
We recommend a concentration of 50 mg/L gentamicin, which is less toxic than standard penicillin/streptomycin solutions.
2.2 stem cells
2.2.1 Preparation Steps - Coating the surface of the culture vessel
If human pluripotent stem cells (hPSCs) or pluripotent mesenchymal/stromal cells are cultured, it is important to treat the surface of the culture vessel with sufficient coating agent. We generally use a coarser extracellular matrix. , such as Matrigel TM.
However, these coatings contain some unclear components such as the substance of the mouse tumor source and the components of the animal source.
If you need to use the coating agent, we recommend using StemAdhre TM, because it is a clear matrix, containing only recombinant protein consisting purely of human sequences, and thus can be considered as an animal-free (ACF), however, is not All tissue culture plates StemAdhre TM Defined Matrix can be used to coat.
The third is from Corning's feeder free, xeno free and chemically defined coating agent Synthemax TM Surface, which is designed to mimic the natural environment of cells, specially treated, ready-to-use coated plastic containers, verified by some hESC Good results have been achieved with the hiPSC cell line. Conversion in the initial transition period to Synthemax TM may need attention, but after a few stages of the cell will restore good growth state.
In serum replacement instructions are not included in the Matrigel TM StemAdhre TM and coating steps.
About Synthemax TM coated plastic containers of detailed information, please visit:
Http://
Key notes:
--XerumFree TM does not contain any growth factors and therefore does not contain bFGF and insulin. When culturing stem cells, we recommend adding bFGF or insulin or IGF.
--XerumFree TM does not contain selenium. When culturing stem cells, we recommend using a medium containing selenium.
2.2.2 Feeder-free culture of hESCs and hiPSCs
DEME coating step according to the above containing 2-3% of the XerumFree TM / F-12 medium in culturing pluripotent stem cells.
The role of external and autocrine signals is matrix remodeling and maintenance of embryonic stem cell turnover (Przybyla, LM and Voldman J. PNAS vol. 109 no. 3, 835-840, 2012). Based on this, in order not to deplete these important factors, it is extremely important to change the procedure to adhere to the medium as described below.
Preparation of hESCs and hiPSCs complete medium
- use conventional basal medium (such as DMEM-F12)
- Add L-Glutamine at a concentration of 2 mM to the medium (eg 1.0 ml of 200 mM mother liquor to a final volume of 100 ml)
- Add bFGF to a final concentration of 4 ng/ml (eg 10 ug/ml of mother liquor to draw 40 ul to a final volume of 100 ml)
- Add 2-mercaptoethanol to a final concentration of 0.1 mM (eg, the mother liquor is 55 mM to absorb 182 ul to 100 ml of medium)
- If an antibiotic protection system is used, we recommend the use of gentamicin at a concentration of 50 mg/ml.
-- Because XerumFree TM does not contain insulin, you can add (recombinant) insulin or IGF to the medium.
- filter the medium if necessary
--Add 2% of XerumFree TM XF205
XerumFree TM medium containing cultured in the first stage hESCs-
- or with Matrigel TM StemAdhre TM coated 6-well plates or culture vessels with Synthemax TM.
- Melt a bottle of fresh hESC or transfer hESC cells from existing media (from feeder or feeder-free culture systems), treat with collagenase or other better methods, use Accutas and sediment as usual, Accutas is non-animal and has protease and collagenase activity, and has shown remarkable effects in the culture of hESC cells.
- or with Matrigel (TM) coated plates StemAdhre TM: Matrigel draw excess coating agent or StemAdhre
--Synthemax TM plate: ready to use without treatment
- Place the cells in complete medium prepared according to the above procedure
-- It is very important to culture the cells by replacing 75% of the culture medium every day for 7 days, leaving 25% of the autocrine-containing medium containing cells.
Note: Compared with MEFs, cells grow better on Matrigel TM package and the board are a higher density, and does not lose its morphological characteristics
Passage of cell culture
-- Wash cells once with DPBS
- Add dispase (for example, add 1 ml of enzyme solution at a concentration of 2 mg/ml per well in DMEM-F12 medium) and incubate at 37 °C
- Disperse cells within 10-15 min by gently tapping one side of the plate
Note: Do not scrape cells
- Transfer the cell-containing dispersed solution to a sterile 15 ml tube. To collect as many cells as possible, rinse the wells with 1 ml of growth medium per well.
- centrifuge and wash twice
- Gently blow the cells with a pipette so that the cells are easily dispersed in the dispase.
Note: Smaller cells and single cells do not survive well.
-- Coating the plates in normal proportions (Matrigel, StemAdhere, Synthemax - see above for instructions)
2.2.3 Cultivation of MSCs
The following protocol is to culture MSCs in a defined environment, either as frozen cell lines preserved in liquid nitrogen or as cultures grown in different cell culture systems.
General consideration
- If MSCs cells are not inoculated in time, they should be stored in liquid nitrogen. Excessive temperature (-80 °C) will cause irreversible damage to the cells.
--Using aseptic technique and operating in a clean bench
- Incubate the cells in a humidified chamber at 37 ° C, 5% CO 2
- The culture vessel must be treated as described in the preparation steps in Part B of the instructions for use on the "coating of the culture surface"
- Cell seeding density is 2000 / cm 2 , avoiding cell fusion of growth, subculture once cell density reaches approximately 70%
- used without serum inactivated by digestive enzymes, such as Accutase TM
- After collection, gently vomit the cells to resuspend, do not vortex cells
- Prepare all materials and equipment used in the cell culture process as described below
- Preheat all solutions and media in contact with cells
Preparation of complete medium
-- You can use the basic medium you routinely use
- Add L-Glutamine at a concentration of 2 mM to the medium (eg 1.0 ml of 200 mM mother liquor to a final volume of 100 ml)
- Add bFGF to a final concentration of 4 ng/ml (eg 10 pg/ml of mother liquor to draw 40 pl to a final volume of 100 ml)
-- If an antibiotic protection system is used, we recommend the use of gentamicin at a concentration of 50 mg/L.
-- Because XerumFree TM does not contain insulin, you can add (recombinant) insulin or IGF to the medium.
- filter the medium if necessary
--Add 2% of XerumFree TM XF205
Thawing cells
During the thawing process, the cells must be carefully handled gently and immediately placed in pre-warmed complete medium.
- Put 10ml of preheated complete medium in a 15ml conical centrifuge tube
-- Transfer cells from liquid nitrogen
- Place the conical flask containing the cells in a 37 ° C water bath and gently stir until all the ice has melted.
-- Immediately disinfect the Erlenmeyer flask with 70% ethanol
- Immediately transfer the cells to a conical flask containing pre-warmed medium and centrifuge at 300 xg for 5 min.
- Pipette the supernatant and carefully resuspend the cells in complete medium
- at 2000-4000 cells / cm 2 cells were seeded at a density in a cell culture dish or Matrigel TM StemAdhere TM coated, or in ready to use Synthemax TM dish (Greiner)
- Incubate cells in a humidified incubator at 37 ° C, 5% CO 2
- The cells are cultured daily by replacing 75% of the medium, and it is very important to retain 25% of the medium containing the autocrine factor.
Subcultured cell culture when approximately 70% confluency is reached
Cell subculture
- Pipette the cell culture medium and wash the cells once with DPBS (no Ca++/Mg++).
- with a sufficient volume of the solution Accutase TM immersing the cell layer and incubated 5min at 37 ℃. If necessary, disperse the cells by gently tapping the sides of the cell culture vessel.
- complete medium the cells were diluted digestion solution (added at least twice the volume of Accutase TM)
- Transfer the cell suspension to a centrifuge tube and centrifuge at 300 xg for 5 min
- discard the supernatant and resuspended the cells by gently aspirating in complete medium
--cell counts
- 2000 cells / cm 2 cell suspension was seeded into a new coated plates or to-use dish Synthemax TM (see above "package culture vessel surface is")
- Incubate cells in a humidified incubator at 37 ° C, 5% CO 2
- Subculture is performed as soon as possible when cell fusion reaches nearly 70%, and is generally subcultured twice a week.
2.3 Primary culture
Primary cell cultures are present in cells that have just been isolated from living tissues or organs. These cells represent the core of the cell culture world: all cell lines to date have been initiated in primary culture. In addition to producing new cell lines, primary culture represents a very important tool, especially in the areas of drug discovery and production, regenerative medicine and basic research.
From a technical point of view, primary cell culture is still the most subtle part of cell culture. The newly isolated cells do not have any selection pressure and retain the characteristics of the internal parts. This is to meet the nutritional and physiological requirements of the newly isolated cells. Ideally, the culture of primary cells should mimic the environment in the body as much as possible, such as extracellular space. This ideal can only be achieved under well-defined cell culture conditions with complete control over the supply of nutrients and cytokines.
Primary cell culture has very different requirements, depending on the source of the tissue. Bovine serum under the condition of all or a derivative thereof specific additives, for example, have proven successful in various XerumFree TM primary cell culture. However, there is no universal formula that satisfies all primary cell type cultures.
The following guidelines are divided into two parts: general recommendations for all cell types and specific requirements for major tissue types.
2.3.1 General recommendations
Regardless of the cell type used, if primary cell culture is selected under serum-free conditions, the following points should be followed.
Serum-free adherent factor
Preparation Steps - Coating the surface of the culture vessel
It is essential to treat the culture surface with sufficient coating agent under well-defined culture conditions. Usually the preparation of extracellular matrix (ECM) is relatively rough, such as mouse sarcoma extract (such as matrigel) or extracted collagen. However, the presence of unclear natural ingredients and animal source ingredients indicates that many applications are problematic.
If you do not want any problems with the animal source ingredients contained in the medium, a quick solution may consider using a cell culture plate that has been treated with a small amount of FBS overnight. This method is convenient and cost-effective, but it will mean taking a step back from the concept of a completely clear culture environment.
Today, ready-made recombinant, well-defined kits can be used, although they are derived from fibronectin, laminin, collagen, E-cadherin, vitronectin and other biosynthetic signal peptides, but can mimic ECM The adherent nature of the protein.
Serum-free enzyme inhibitor
Digestive enzymes
There are two main ways to start a primary culture: a product that is isolated or enzymatically dissociated from a tissue block. In the latter method the starting tissue is digested with a proteolytic enzyme or enzyme solution, such as dispase, collagenase and trypsin.
Any proteolytically active ingredient must be carefully neutralized/inactivated prior to seeding the cells. Especially when using trypsin, this problem must be solved.
The use of standard trypsin preparation in a serum-free environment can be problematic because the serum contains trypsin inhibitors. After dispersing the cells, trypsin activity must be inactivated under serum-free conditions, and a highly potent trypsin inhibitor such as soybean trypsin inhibitor can be used.
As an alternative to trypsin, it is strongly recommended Accutase TM, because it need not be inactivated. This recombinant non-mammalian-derived enzyme has been efficiently used in a range of primary cell cultures, including primary smooth muscle cells, primary human endothelial cells, and primary chicken nerve cells.
Serum-free protein binding
Use of antibiotics
Antibiotics bind to serum plasma proteins, especially albumin fragments, like other compounds. Therefore, the same antibiotic concentration will show higher biological activity under serum-free and albumin-free conditions, and the increased activity will have a detrimental effect on cell growth. In particular, streptomycin interferes with the level of protein synthesis in mammalian cells.
If antibiotic-free culture is not feasible, we recommend the use of gentamicin at a concentration of 50 mg/L.
2.3.2. Specific recommendations for cell types
 Primary cell cultures have different cell culture requirements depending on the tissue source.
In this booklet we do not specifically describe the primary cell culture protocol because different cell types are distinct. Generally we recommend the isolation of primary cells by conventional methods, and washed with 5-fold concentrated serum XerumFree TM replaced.
This will meet the nutritional needs of most cell types. In fact, in the field of mammalian cell culture, the nutritional needs are slightly different and more dependent on cell types, such as hepatocytes requiring higher nutrient concentrations.
The demand for growth factors and hormones varies among different types of cells.
The following table lists the recommended preparing a cell culture medium instead of animal serum with the XerumFree TM.
With regard to the four cell types specified, the addition of growth factors and hormones is ideal for cell development and proliferation.
2.3.3 Establishment of recommended cell culture media for four major primary cell types
* In order to make the cells adhere well and spread, we strongly recommend CaCl 2 (0.06 mM)
Nb. Do not filter XerumFree TM . We recommend the preparation of glutamine-containing basal medium, if required growth factors and hormones was filtered off, then add XerumFree TM may then be filtered off.
Nb. XerumFree TM is concentrated 5-fold, 5-fold so the concentrate was added FBS volume lower than the commonly used (such as 10% FBS equivalent to 2% XerumFree TM).
Nb. For other primary cultured cell types, TNC Bio can be contacted, and we will provide maximum support for the recommended amount of growth factors and hormones.