Question

6. How did scientists succeed in culturing normal early mouse embryonic cells? Describe the experimental procedures and the methods they used in the pluripotency tests of the cultured cells.

Answer 1

• Injecting stem cells into mouse embryos is standard practice to examine how well the cells incorporate and aid in tissue development, but previous attempts to get human stem cells to contribute normally to embryo development have failed. Reporting in Cell Stem Cell last week (December 17), researchers from the University of Cambridge successfully implanted human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) into developing mouse embryos and observed them becoming all types of tissue layers.
• The University of Cambridge-based authors suggest that their recent success comes from their decision to inject the stem cells at a later stage of embryo development. The hiPSCs and hESCs “have the capacity to participate in normal mouse development when transplanted into gastrula-stage embryos,” they wrote in their study.
• The human stem cells differentiated into each of the three primary tissue layers—endoderm, ectoderm, and mesoderm—of the developing mouse. The researchers found no difference between the differentiation abilities of hiPSCs and hESCs. “We conclude that transplanted [human pluripotent stem cells] and their progeny proliferate and contribute normally to the developing embryo, irrespective of transplant stage, site, or cell type,” the authors wrote.
• The mouse embryos were cultured for two days, not long enough for differentiation into final tissue types—but the embryos appeared normal during this time period. “The Cambridge team has shown definitively that when stem cells are introduced into early mouse embryos under the right conditions, they multiply and contribute in the correct way to all the cell types that are formed as the embryo develops,” Jeremy Pearson, Associate Medical Director at the British Heart Foundation, which helped fund the study, said in a press release.

“Our finding that human stem cells integrate and develop normally in the mouse embryo will allow us to study aspects of human development during a window in time that would otherwise be inaccessible,” study coauthor Victoria Mascetti said in the press release.

Experimental procedures

• Stem cell research is a rapidly expanding field with the potential to develop therapeutic agents to treat diseases as well as study disease development from early stages. The culture of human pluripotent stem cells shares many of the same protocols as standard mammalian cell culture.
• However, the successful culture and maintenance of human pluripotent stem cells (hPSCs) in an undifferentiated state requires additional considerations to ensure that cells maintain their key characteristics of self-renewal and pluripotency.
• There are several basic techniques needed for the culturing of mammalian cells, including thawing frozen stocks, plating cells in culture vessels, changing media, passaging and cryopreservation.
• The protocols in this document represent a subset of the standard operating procedures used to maintain and culture stem cells at the Massachusetts Human Stem Cell Bank, and have been thoroughly testing and verified.

1. A Stem cell culture considerations

• Stem cell research is a rapidly expanding field with the potential to develop therapeutic agents to treat diseases as well as study disease development from early stages. However, to fulfill this promise, researchers need to have access to standardized protocols for the development, maintenance and differentiation of these unique cells. Such “best practices” will allow comparisons of different studies and hasten the refinement of these techniques.
• Such standardization can be driven by resources such as StemBook and by stem cell banks. In addition to standardization of these best practices, stem cell banks also serve as valuable resources of properly identified, quality controlled and characterized cell lines as well as helping to navigate the legal and IP issues that are common to working with stem cells.

The protocols in this, represent a subset of the standard operating procedures used to maintain and culture stem cells at the Massachusetts Human Stem Cell Bank, and have been thoroughly tested and verified.

Successful stem cell culture

• The culture of human pluripotent stem cells shares many of the same protocols as standard mammalian cell culture.
• However, the successful culture and maintenance of human pluripotent stem cells (hPSCs) in an undifferentiated state requires additional considerations to ensure that cells maintain their key characteristics of self-renewal and pluripotency.
• Successful hPSC culture requires the recreation of the in vivo stem cell microenvironment, or “niche”, which includes growth factors, cell-to-cell interactions and cell to matrix adhesions. Unlike many cell types, hPSCs are grown in aggregates, or colonies, which helps create this niche.
• Standard culture of hPSCs involves exposure to media enriched with growth factors found in fetal bovine serum (FBS) or defined serum replacements. In addition, standard hPSC culture systems utilize support cells such as an inactivated mouse embryonic fibroblast (MEF) feeder layer to support growth and prevent differentiation.
• These cells provide necessary intercellular interactions, extracellular scaffolding and factors creating a robust and stable hPSC culture environment.

There are several basic techniques needed for the culturing of mammalian cells, including thawing frozen stocks, plating cells in culture vessels, changing media, passaging and cryopreservation. Passaging refers to the removal of cells from their current culture vessel and transferring them to one or more new culture vessels. Passaging is necessary to reduce the harmful effects of overcrowding and for expansion of the culture. Protocols provided in this manual describe the standard culture of hPSCs. These procedures have been shown to be reliable and generate reproducible experimental data.

Although the standard culture protocols described here use a variety of animal products, any clinical use of hPSCs will require elimination of these products as they pose a risk of exposure to retroviruses and other pathogens from the culture environment. Many approaches have been published for culturing hPSCs in an entirely animal-free environment, including the use of human fibroblasts and serum, the use of defined substrates, and replacement of serum with defined growth factors.1-3

2 Quality control of cell cultures

It is essential that researchers ensure the sterility, authenticity and genetic stability of cell lines used in their work in order to publish and provide reproducible and informative experimental data. Upon receipt of a new cell line, it is highly recommended that cells assayed for the criteria outlined below and monitored at regular intervals to confirm these characteristics

3.Cell sterility

• Cells in continuous culture are generally vulnerable to microbial contamination. Bacterial and fungal contamination can cause cell death and eventual loss of entire cultures. Human stem cell cultures are particularly susceptible since they are commonly cultured in enriched media without antibiotics.
• It is highly recommended that cells received from any provider should be tested for microbial contamination before initial use and at regular intervals during routine culture in the laboratory. Since microbes are everywhere in the culture environment, stringent practice of aseptic technique is essential for preventing culture contamination.

In addition to bacteria and fungi, another common contaminant is mycoplasma. These intracellular microorganisms are generally smaller than bacteria and may affect cell growth, particularly at high levels of contamination. However, persistent infections of cells can result in genetic and phenotypic changes. Common sources of mycoplasma include contaminated materials of animal origin such as serum, trypsin and primary feeder cell cultures. Thus it is important to test for the presence of mycoplasma on a regular basis and discard contaminated cultures. Common methods used to detect mycoplasma include enzymatic assays, polymerase chain reaction (PCR), culture in selective media, and DNA staining of test cells to visualize mycoplasma that grow in close association with the cell membrane. Although the least sensitive, DNA staining with 4′6-Diamidino-2-phenylindole (DAPI), a fluorescent stain that binds strongly to DNA, is a simple method that can be employed in most laboratories to detect mycoplasma. Commercially available kits are offered by a number of reagent companies.

Viruses are another form of contamination. Viruses can alter a cell line's characteristics to varying degrees through multiple activities. These include utilization of host cellular resources for viral replication and integration into the host genome. Viral infection may affect experimental data and could result in misleading interpretations. In addition, cultures that are contaminated with blood borne viruses capable of human infection pose a serious health risk. Common sources of viral contamination include animal products and preparations such as bovine serum, antibodies and mouse embryonic fibroblasts.6,7 Although testing for viral contamination is conducted by stem cell banks and repositories on a regular basis, this testing is not common practice for individual research laboratories. It is recommended that laboratories test cells for viral infection prior to their distribution and that recipient laboratories request documentation of this testing. Several companies offer a wide range of viral testing services; their fees depend on the breadth and types of tests performed.

1.2.2. Cell line authenticity

Cells in culture are at high risk for cross-contamination since most laboratories routinely culture multiple cell lines simultaneously. Studies have shown that up to 30% of cell lines donated to public repositories are contaminated by rapidly growing cell types7,8 as was the case with HeLa cells, which can outgrow and replace the original cell lines. This cross-contamination can be more subtle and lead to false data and misleading conclusions. This is of particular interest in regulated environments where authenticity or identity is a key component of quality controls systems for clinical applications.

Authentication of cell lines obtained from outside sources prior to their use in experiments is essential and can be achieved by comparing the unique features of received cell lines against those of the original isolate. Several approaches used to authenticate cell lines. Genotyping takes advantage of the small genetic variations between individual cell lines. Current DNA typing employs PCR-based techniques to analyze similar hypervariable satellite DNA sequences and single nucleotide polymorphisms. Multiple companies offer genotyping services for a small fee.

1.2.3. Cell line stability

• Cells grown in culture have a tendency to accumulate genetic and/or phenotypic changes. Studies have shown that long-term culture of hPSCs result in genetic abnormalities including changes at the chromosomal level, which can be detected by karyotyping methods. The karyotypes of human pluripotent stem cell cultures should be frequently tested by a certified cytogenetics laboratory.
• Multiple techniques are used to ensure that pluripotent stem cell lines retain their stem cell phenotype, and include expression of stem cell markers, both molecularly and as expressed intracellular or surface markers and the ability to form the three embryonic germ layers.

2. Reagent preparation

Several reagents must be prepared prior to maintaining human pluripotent stem cells (hPSCs) in culture. It is important to have a firm grasp of the characteristics of each reagent, including components, appropriate preparation, storage and shelf life. This chapter details the preparation of the following reagents necessary for hPSC culture:

0.1% Gelatin Solution

Inactivated MEF Medium

Basic Fibroblast Growth Factor (bFGF) Solution

hPSC Pluripotent Culture Medium

Collagenase Solution

Many reagents, especially Knockout Serum Replacer (KOSR), bFGF, and collagenase can have significant variation between lots. When working with a new lot, it is important to compare it directly to the lot that is currently in use, to ensure that the current culture quality and viability are maintained.

2.1. B1 Preparation of 0.1% gelatin solution

2.1.1. Supplies

500 ml sterile glass bottle

Weigh boat

2.1.2. Reagents

Gelatin, Porcine

Endotoxin-free

2.1.3. Procedure

2.1.3.1. Prepare 0.1% gelatin solution

Note: Adjust the volume of water and gelatin powder proportionally if a volume other than 500 ml of gelatin is prepared.

Add 0.5 g of gelatin powder to a clean 500 ml pyrex bottle.

Add 500 ml endotoxin-free (e.g., MilliQ) water to the bottle.

Swirl to mix. (At this stage, the gelatin is not soluble).

Autoclave for 30 minutes within 2 hours after mixing.

Cool the 0.1% gelatin solution to room temperature and store at 4–8°C until use. Use this solution within two months of preparation.

2.2. B2 Preparation of iMEF culture medium

2.2.1. Supplies

5 ml sterile serological pipets

10 ml sterile serological pipets

25 ml sterile serological pipets

500 ml bottle connected with a 0. 22 μm Stericup

2.2.2. Reagents

DMEM-liquid (Invitrogen 11965-118)

MEM Non-Essential Amino Acid Solution (NEAA) (Invitrogen 11140-050)

Heat Inactivated Fetal Bovine Serum (HI-FBS) (Invitrogen 16000-069)

70% ethanol (Diluted from 95% ethanol, Fisher NC9608803)

2.2.3. Procedure

2.2.3.1. Preparation of iMEF culture media

In the hood, open a 500 ml filter and bottle unit. Label the bottle:

iMEF and date of preparation

Expiration date (mm/dd/yyy, 14 days after media preparation). For example: Exp: 07/10/2008

Your initials

Add the appropriate amount of ingredients to the 500 ml filter cup as shown in the table below:

Note 1: The DMEM may be measured by pouring directly into the graduated filter cup of the filter/bottle unit. The other solutions should be added using serological pipets.

Note 2: Scale up or down proportionally if another quantity of medium is needed.

Filter the media through the Stericup filter into the attached bottle.

Store the medium bottle at 4°C, and use the medium within 14 days.

2.3. B3 Preparation of basic fibroblast growth factor (b-FGF) stock solution

2.3.1. Supplies

1.5 ml or 2 ml sterile microcentrifuge tubes

1000 μl sterile pipette tips

10 ml sterile serological pipets

25 ml sterile serological pipets

50 ml sterile centrifuge tubes

70% ethanol spray

Forceps

2.3.2. Reagents

Basic Fibroblast Growth Factor (b-FGF) (Invitrogen PHG0021)

PBS with 0.01% CaCl2 and 0.01% MgCl2 (Invitrogen 14040-141)

30% BSA (Bovine Serum Albumin) (Sigma A9576)

2.3.3. Procedure

2.3.3.1. Preparation of 0.1% bovine serum albumin (BSA) solution

Add 0.1 ml of the 30% BSA to 30 ml of PBS.

Cap the tube and mix the solution by inverting 3–4 times. It is now a 0.1% BSA solution.

2.3.3.2. Preparation of 10 ug/ml b-FGF stock solution

Place one vial of 100 μg lyophilized b-FGF in a 50 ml tube and briefly centrifuge the b-FGF vial at 200 × g for 5 minutes to bring the lyophilized b-FGF to the bottom of the vial.

Use a pair of forceps to carefully remove the vial from the 50 ml tube.

In a biosafety cabinet, carefully remove the cap gently. Avoid contact with any b-FGF powder that has stuck to the cap.

Remove 500 μl of 0.1% BSA solution and add it to the b-FGF vial (Do not touch FGF pellet at the bottom).

Carefully replace the cap.

Carefully remove the cap and gently pipette up and down a few times.

Transfer the b-FGF solution to the 50 ml tube.

Rinse the b-FGF vial with 500 μl solution from the “FGF” tube to collect any residual b-FGF protein in the vial. Return the solution back to the 50 ml tube. Repeat this 2–3 times.

2.3.3.3. Aliquots of b-FGF (10 ug/ml) stock solution

In the hood, place a set of 50 sterile 1.5 ml or 2 ml vials in a cryo-vial holder.

Aliquot the solution.

Store at −80ºC.

2.4. B4 Preparation of pluripotent stem cell culture medium

2.4.1. Supplies

1000 μl sterile pipette tips

20 μl sterile pipette tips

5 ml sterile serological pipets

10 ml sterile serological pipets

25 ml sterile serological pipets

50 ml sterile centrifuge tubes

250 ml Stericup Filter

500 ml Stericup Filter

70% ethanol spray

2.4.2. Reagents

DMEM-F12 media (Invitrogen 11330-032)

Knockout Serum Replacer (KOSR) (Invitrogen 10828-028)

L-glutamine, non-animal, cell culture tested (Sigma G-8540)

MEM Non-Essential Amino acid solution (Invitrogen 11140-050)

Basic Fibroblast Growth Factor (b-FGF); Stock 10 ug/ml

beta-Mercaptoethanol (Sigma M7522)

2.4.3. Procedure

2.4.3.1. Preparation of culture medium (CM) preparation

Note: Please note that many iPS culture media formulas suggest the inclusion of antibiotics. When working with any cell line, check with the cell line provider for specific media recommendations.

Note: If the final total volume of medium is different from the one listed in the table below, adjust the volume of ingredients proportionally.

In a biosafety cabinet, open a 250 ml or 500 ml filter and bottle unit.

Label the bottle of the filter unit (Culture Medium and date of preparation, expiration date (14 days after media preparation), initials)

According to the final total volume to be prepared, add appropriate amounts of ingredients to the 250 ml or 500 ml filter unit cup as shown in the table below.

Note: The DMEM-F12 may be measured by pouring directly into the graduated filter cup. Other reagents should be added with sterile serological pipets.

Note: When thawing cell lines that have been previously cultured under different conditions, in most cases, it is possible to adapt the cells to the culture medium described here by thawing the cells into this formulation. Thawing hPSCs is fully described in the protocol;C3 Thawing and Seeding, and replacement of medium for Pluripotent Stem Cells.

Filter the media through the 0.22 μM filter.

Store the medium bottle at 2–8°C, and use the medium within 14 days.

2.5. B5 Preparation of collagenase solution

2.5.1. Supplies

Weigh boat

25 ml sterile serological pipets

150 ml Stericup filter

70% ethanol spray

2.5.2. Reagents

DMEM-F12 media

Collagenase Type IV

2.5.3. Procedure

Note: The following procedure is to prepare a collagenase solution at a concentration of 1 mg/ml.

Weigh 100 mg of Collagenase Type IV powder into a weigh boat.

Using a 25 ml pipette, transfer 20 ml of DMEM/F12 medium to the weigh boat containing the collagenase.

Pipette DMEM/F12 medium up and down in the boat to dissolve the collagenase powder. The collagenase should dissolve almost instantly.

When the collagenase is completely dissolved, transfer the solution to a 50 ml tube.

Rinse the residual collagenase in the boat with an additional 20 ml of DMEM/F12 medium and add this to the 50 ml tube containing the collagenase solution, tighten cap.

Spray the 50 ml tube containing the collagenase solution with 70% ethanol, and place the tube in the biosafety cabinet.

To a 150 ml 0.22 μm filter unit pour 60 ml of DMEM/F12 medium and to this add the 40 ml of collagenase solution from the 50 ml tube.

Filter sterilize the collagenase solution (1 mg/ml).

Store the solution at 4°C, and use within 14 days.

2.5.3.1. References and Suggested Reading

Freshney, RI. Culture of Animal Cells: A Manual of Basic Technique. 5th edition. New Jersey: John Wiley & Sons; 2005. P 73–85

Massachusetts Human Stem Cell Bank. 2009. Standard Operating Procedures. Available at http://www.umassmed. edu/mhscb.

3. C Cell culture

The protocols provided here describe the standard culture techniques used for pluripotent stem cells. The protocols discussed here have been shown to be reliable and generate reproducible experimental data. The protocols include:

Gelatin Coating of Culture Plates

Thawing and Seeding of Frozen Inactivated Mouse Embryonic Fibroblasts (iMEFs)

Thawing and Seeding of Pluripotent Stem Cells onto a Mouse Embryonic Fibroblast (iMEF) Feeder Layer

Replacement of Medium for Pluripotent Stem Cell Culture

Passaging of Pluripotent Stem Cells on Fresh Mouse Embryonic Fibroblast (iMEF) Plates

Harvesting and Cryopreservation of Pluripotent Stem Cells

3.1. C1 Gelatin coating of culture plates

3.1.1. Supplies

6-well tissue culture plates

5 ml sterile serological pipettes

10 ml sterile serological pipettes

3.1.2. Reagents

0.1% gelatin solution

3.1.3. Procedure

3.1.3.1. Coat culture plates using 0.1% gelatin solution

Warm to room temperature an appropriate amount of gelatin solution. For 6-well plates, use 2 ml of 0.1% gelatin solution for each well.

Place the plates that are to be coated in the biosafety cabinet.

Label the cover of the plate (not over the wells) with: “G” for gelatin, date, initials

Add 2 ml of 0.1% gelatin solution to each well.

Tilt or swirl the plates in several directions so that the liquid covers the entire surface area.

Place the plates in a 37°C incubator.

Note: The plates will be ready for use in 4 hours. They can be used for up to 7 days.

3.2. C2 Thawing and seeding of frozen inactivated mouse embryonic fibroblasts (iMEFs)

3.2.1. Supplies

5 ml sterile serological pipettes

10 ml sterile serological pipettes

25 ml sterile serological pipettes

15 ml sterile centrifuge tubes

50 ml sterile centrifuge tubes

70% ethanol spray

3.2.2. Reagents

iMEF culture medium

Gelatin-coated plates

Frozen iMEFs

3.2.3. Procedure

3.2.3.1. Prepare gelatin plate(s) in the biosafety cabinet

Remove gelatin plate(s) from the incubator and place inside the biosafety cabinet.

Aspirate the gelatin solution from the plate(s) completely with a Pasteur pipette.

Return the plate(s) to the incubator for later use.

3.2.3.2. Thaw iMEFs

Note 1: To seed 6 wells in a 6-well plate, 1.2–1.5 × 106 iMEFs are required (assuming a 90% recovery of iMEFs from one freezing and thawing cycle).

Note 2: Thaw no more than two vials at a time.

Note 3: Wear eye protective safety glasses and insulated gloves when removing cryovials from the liquid nitrogen freezer.

Bring an appropriate amount of MEF medium (iMEF CM) to 37°C (assuming 2 ml per well + 9 ml for thawing)

Place the warmed iMEF CM in the biosafety cabinet after thoroughly cleaning the outside of the container with 70% ethanol.

Label a sterile tube (both on the cap and side) in the biosafety cabinet as “iMEF”.

Transfer 9 ml iMEF CM into the “iMEF” tube.

Open the LN2 freezer and take out the correct vial(s) of iMEFs.

Warm the vial(s) slightly between gloved hands while walking to the water bath.

In the 37°C water bath, immerse the vial in the water without submerging the cap. Swirl the vial gently. After about 30 seconds, check frequently to see if the frozen solution is beginning to melt. This process may take up to 1 minute.

When there is only a small piece of ice floating in the vial, remove the cryovials from the water bath.

Place the vial(s) in the biosafety cabinet after thoroughly cleaning the outside of the cryovials with 70% ethanol.

In the biosafety cabinet, pipette the cells up and down gently a couple times, and then transfer the cell suspension drop-wise to the tube labeled “iMEF” while gently swirling the tube. This will help to reduce osmotic shock to the cells.

Centrifuge for 5 minutes at 200 × g.

Return the 50 ml centrifuge tube to the biosafety cabinet after spraying it with 70% ethanol.

Aspirate the supernatant from the tube. Be careful not to touch the cell pellet.

Gently flick the bottom of the tube to loosen the cell pellet.

Add 10 ml of iMEF CM to resuspend and mix the cells by pipetting gently up and down a few times.

If necessary, count iMEFs.

Add iMEF CM to the iMEF tube for a final cell concentration of 0.1–0.13 × 106 iMEFs/ml. Image shows correct concentration of MEFs.

MEF cells at confluence of 0.1–0.13 × 106 iMEFs/ml. Bar represents 1000 μm.

3.2.3.3. Seed iMEFs in 6-well plate

Take the gelatin plate(s) from the incubator to the biosafety cabinet. Label the plates with “iMEF”, date and initials.

Resuspend the iMEFs completely by gently pipetting up and down a few times and add 2 ml/well to gelatin coated wells.

When all plates in the biosafety cabinet are seeded, slide the plates back and forth and side to-side (cross motion) three to five times inside the biosafety cabinet to evenly distribute cells and make another 3–5 cross-motions with the plates when transferred to the incubator.

3.2.3.4. Post seeding

Do not disturb the freshly seeded plates for at least the next 12 hours.

Check each plate of cells under the microscope the following day for MEF quality and for signs of contamination

Record observations

Repeat the above each day until the plates are used for pluripotent stem cell culture.

Note: Discard unused or contaminated iMEF plates after 5 days

3.3. C3 Thawing and seeding, and replacement of medium for pluripotent stem cells

3.3.1. Supplies

5 ml sterile serological pipettes

10 ml sterile serological pipettes

25 ml sterile serological pipettes

15 ml sterile centrifuge tubes

50 ml sterile centrifuge tubes

70% ethanol spray

3.3.2. Reagents

DMEM-F12

hES Cell Culture Medium

iMEF plate containing iMEFs

3.3.2.1. Monitor iMEFs under microscope

Observe the number of plates needed for this protocol one under a microscope.