PROTOCOLFastGroTM

Protocol for using FastGroTM,
a fully defined, animal-component freecell culture supplement, to replace FBS
Performing cell culture without serum can be challenging. However, the rewards largely compensate for the efforts, andre-discovering the basics of cell culture quickly develops into a passion. The intention of this paper is to guide the user through asmooth transition to serum-free conditions while avoiding inadequate or wasted efforts.
Ideally, the transition to serum-free conditions should be carried out over several passages to gradually select cells that can growunder serum-free conditions. However, direct adaptation to serum-free environments are often successful, provided all crucial
aspects are addressed properly. Regardless of the method used, key concerns include the growth state of the cellular inoculum, cellseeding density, sub-cultivation techniques, and biophysical attributes of the cell culture system.
MP Biomedicals’ FastGro™ serum replacement is an effective FBS supplement and has been designed for use in the same manneras conventional cell culture sera.

IMPORTANT NOTES BEFORE USING CONCENTRATED FastGro™:
FastGro is a cell culture medium additive replacing serum; it is not to be used as a final medium.
FastGro must be added to a basal cell culture medium (e.g. IMDM, DMEM-F12 or any other basal medium of choice).
FastGro does not contain growth factors such as cytokines, hormones, etc, and also lacks insulin*.
*The option to add insulin (or additional growth factors) is based on user requirements. If insulin is needed for cell growth and performance, weadvise adding recombinant insulin at a concentration of 1.25 mg/L final cell culture medium.

CONTENTS:

1. PREPARING
1.1 General preparation of the cell culture medium
1.2 General adaptation methods to serum-free conditions
1.2.1 Direct adaptation

2. USING FastGro
2.1 Cell Lines
2.1.1 Anchorage-dependent cell lines
2.1.2 Anchorage-independent cell lines

1.2.2 Sequential adaptation

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PROTOCOLFastGroTM

1Preparing FastGroTM
1.1 GENERAL PREPARATION OF SERUMFREE CELL CULTURE MEDIUM
Gently shake the bottle of FastGro™ shortly before use.
Add FastGro™ to your preferred basal medium at thesame concentration as for serum (e.g. 10%).
Do not add any antibiotics at this stage. Antibiotics, likemany compounds, bind to the plasma proteins of serum,in particular to the albumin fraction. Thus, the same
concentration of antibiotics will exhibit a much higher
biological activity in serum- and albumin-free conditions,potentially impacting cell growth.
In instances where ‘antibiotic-free culture’ is deemed
unworkable, the use of gentamycin is suggested at aconcentration of 50 mg/L.

1.2 GENERAL ADAPTATION METHODSTO SERUM-FREE CONDITIONSThere are essentially two approaches to adapt cells togrowth in a serum-free environment:
1.2.1 Direct Adaptation
This is carried out by a direct transfer of the cells
from the serum-containing medium into the serumfree medium.
1.2.2 Sequential Adaptation or Weaning Method
Pass the cells from the original serum containing medium
sequentially through phases where each step halves theserum-supplemented media, thus increasing the serumfree media to the following aproximate values:

Phase 1:
50% FastGro-supplemented medium/
50% Serum-supplemented medium
Phase 2:
75% FastGro-supplemented medium/
25% Serum-supplemented medium
Phase 3:
87.5% FastGro-supplemented medium/
12.5% Serum-supplemented medium
Phase 4:93.75% FastGro-supplemented medium/
6.25% Serum-supplemented medium
Phase 5:96.88% FastGro-supplemented medium/
3.12% Serum-supplemented medium
Phase 6:98.44% FastGro-supplemented medium/
1.56% Serum-supplemented medium
Phase 7:
100% FastGro-supplemented medium
If reduced growth is observed, go back one step andcontinue after growth is reestablished.
Cell cultures may consist of cell lines (adherent orsuspension growth) or primary cultures. Moreover,from a functional standpoint, cell types may bedifferentiated to various degrees or exhibit
undifferentiated characteristics, as in the case ofstem cell preparations. In each instance, the adaptation
protocol must take into account the specificrequirements of the cell type to guarantee the best
chances for success.

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PROTOCOLFastGroTM

2Use of FastGroTM

Our preference is to omit trypsin and instead use
Accutase™ or Detachin™ to dissociate the cellmonolayer. These cell detachment solutions have beendeveloped to meet the most demanding requirements for
gentle and effective detachment of adherent cells. Cell
membranes and surface epitopes will not be harmed
during detachment, and the structural and functional
quality of the surface proteins will remain intact.

2.1 CELL LINES
The following protocols are valid for normal (diploid,limited lifespan), transformed or immortalized cell lines(with indefinite lifecycle).
2.1.1 Anchorage-dependent Cell Lines
Critical success factors:
coating of the cell culture support for
optimal cell attachment

CSeed cells at 20,000 cells per cm2 in complete medium
as prepared under point 1
It is important to observe a high seeding density during
the first steps of the adaptation process. Cells normallysecrete a host of factors into the culture medium that
control cell attachment, growth and proliferation.
However, these factors are absent in the fresh serum-free
medium during the seeding step. A critical level of celldensity is essential to induce immediate and sufficient
production of these autocrine/paracrine factors.

DIncubate and maintain the cell cultures at 37°C until
they reach 80-90% confluency.
During this period, change 75% of the medium every 2-3days. Do not discard the spent medium. Instead, harvest
the conditioned medium, sterile filter and put aside at
4°C for use in subsequent steps. If the cells appear
stalled at any point, allow them more time to adapt totheir new, serum-free environment.

E

When near confluency is reached, split the cells at a
1:2 or 1:3 ratio.
For the second passage in FastGro™, a coating is not
required, but use of conditioned medium is strongly
suggested. This medium fraction contains autocrine
factors that regulate attachment, spreading, growth andproliferation. Seed cells in a mixture consisting of 75%
fresh medium and 25% conditioned medium, collectedduring the previous passage. Continue supplying cellswith 75% fresh medium every 2-3 days and collect theconditioned medium as described in step D above.

minimize action of trypsin
choice of the antibiotic system
Experimental Steps:
A

Coat the cell culture surface with an adequate
cell-attachment factor by using:a commercial coating kit such as Pronectin™ F,
MapTRIX™ or equivalent.
a Fibronectin or Poly-L-Lysine coating, or a small
quantity of FBS (e.g, 500 μL for a T25 flask) withovernight incubation at 37°C, followed by two
washes with PBS or fresh medium.
ready to use plastics that provide an improved
attachment of adherent cells.

B

Dissociate the cell monolayer
The use of standard trypsin preparations can become
somewhat problematic in the absence of serum, whichcontains trypsin inhibitors. It is therefore important tominimize the proteolytic activity of residual trypsin inserum-free conditions to avoid irreversible damage to thecells. This is best achieved by utilizing trypsin inhibitors(e.g. from soybean) or by employing a non-mammaliandissociation reagent, such as Accutase™, which does
not require inactivation or removal during passaging.
Alternatively, an additional wash step of the cell pellet
will remove the majority of the residual trypsin. However,this procedure employs an extra centrifugation step thatcan be damaging for some cell types.

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PROTOCOLFastGroTM

FRepeat step EE until the cells exhibit growth dynamics
comparable to their former growth in serumsupplemented medium.
At this point, the cell line can be considered fully adapted.
This may take up to a total of 4-6 passages.

GFrom this point on, antibiotics may be added to theculture medium.
We advise on the use of the large-spectrum antibiotic
gentamicin; this antibiotic has a significantly reduced
cytotoxicity as compared to the standard Penicillin/
Streptomycin cocktails. The suggested concentration ofgentamicin is 50 mg/L.

HC

Resuspend the cell pellet in FastGro™ supplemented
medium at a density of 106 cells/mL.
It is important to observe a high seeding density during
the first steps of the adaptation process. Cells normallysecrete a host of factors into the culture medium that
control cell growth and proliferation. However, during theseeding step, these factors are absent in the fresh serumfree medium. A critical level of cell density is essential
to induce immediate and sufficient production of these
autocrine/paracrine factors.

DIncubate and maintain the cell cultures at 37°C until theyreach a density of approximately 3-5x106 cells/mL.

ESplit the suspension cultures at a 1:3 or 1:4 ratio byadding an appropriate volume of fresh medium(e.g. 25 mL of cell suspension and 75 mL FastGro™
supplemented medium, to be dispatched into 4
separate culture vessels)

F

Repeat step EE until the culture exhibits growth dynamics
as originally observed in serum-supplemented medium.
At this point, the cell line can be considered fully adapted
and may be split at the original ratios during serumsupplemented culture.

GAntibiotics may now be added to the culture medium.
We suggest the use of gentamicin at a concentrationof 50 mg/liter; this antibiotic has a much lower
cytotoxicity as compared to the standard Penicillin/
Streptomycin cocktails.

Once adapted, the original split ratio (in serumsupplemented conditions) may be applied.
2.1.2 Anchorage-independent Cell Lines
The following protocol is adjusted for cell lines that
already grow in suspension. For the adaptation ofadherent cells to FastGro™ suspension growth, pleasesee MP Bio’s Application Notes for FastGro.
Critical success factor:
Choice of the antibiotic system
Experimental Steps:

A

When cell densities of 3-5x106 cells/mL are reached(depending on the cell line) start switching to FastGro™
supplemented medium.
Harvest the cell suspension, take out a small aliquot forcell counting and centrifuge the entire suspension at
200 x g for 5 minutes.

B

Perform a cell count according to your standard
laboratory procedures.

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