THE MAIN ASPECTS OF ESTABLISHING A STEM CELL LABORATORY

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THE MAIN ASPECTS OF ESTABLISHING A STEM CELL LABORATORY

Human stem cells are unique as they undergo self renewal and differentiation to become specialized cells once given the right signals. The promise of stem cell research in understanding the cell biology, regenerative medicine and as drug screening has made this research an exciting area to explore. Hence, despite its ethical issues, stem cells are continuously being studied by establishing more stem cell laboratories. However, to produce robust, reproducible and reliable data, several important aspects must be reviewed and can be done by visiting established laboratories and getting insights from experienced users. This is to ensure the efficiency and effectiveness of the culture and experimentation and to prevent waste (Inamdar et al., 2012). The following discussions are describing about the basic human embryonic stem cell (hESC) academic laboratory with the final material is not intended for human application and with involvement of animal in vivo studies.

There are two main aspects to be considered in establishing a stem cell laboratory which can be categorized into administrative plan and physical plan. Administrative plan involves establishing protocols, seeking approvals, reporting and documentation and meanwhile physical plan involves designing and layout of the laboratory, equipment and personnel (Wesselschmidt & Schwartz, 2013). Stem cells derived from human embryos are facing with ethical issues hence permissions and approvals must be granted before starting the research. In the United Kingdom, there is a system called Integrated Research Application System (IRAS) to seek relevant approvals from HFEA, Research Ethic Committee (REC), and NHS & R&D Office in a single application process as shown in Table 1. As there will be animal in vivo studies, the studies must be conducted under licence from Home Office. Documentation including developing protocols is important to be recorded in laboratory note books. Protocols can be deviated from standard procedures and the version of a protocol is used must be mentioned so that the best technical procedures or the effect of changing procedures can be traced. If any legal conflict arises, laboratory note books are acceptable evidences which keeping records of all original work and fail to do so can even cause retraction of publications. It is helpful to document the procedures in organized and structured manner such as typical procedures shown in Table 2. In fact, this good scientific practice promotes efficient use of research resources (Inamdar, et al., 2012).

Regulator

Description

HFEA

  • The independent regulator for IVF treatment and embryo research
  • To provide information to the public and policy makers about the research
  • To license and monitor human embryonic research
  • Regulates the storage of embryos

REC

  • To safeguard the rights, dignity and welfare of people involved in the health research
  • Laboratory research that uses embryonic cell line(s) requires REC Favourable Opinion

NHS & R&D Office

  • An additional to ethical approval
  • Must get the permission before the studies start
  • To claim insurance/indemnity

Table 1. Regulators to seek for permissions and approvals related to human embryonic stem cells in the United Kingdom. Adapted from UK Stem Cell Tool Kit http://www.sc-toolkit.ac.uk/regulatoryroutes.cfm

Documentation

  • Reception of cell lines
  • Passaging stem cells
  • Preparation of cultural media
  • Preparation of feeder cells
  • Preparation of stem cell lines for analysis
  • Cryopreservation of stem cell lines in liquid nitrogen store
  • Thawing stem cell lines from liquid nitrogen store

Table 2. Some of specified documented protocols in the stem cell culture laboratory. Modified from (Inamdar, 2012).

The scope of work such as the numbers and types of cell lines to be cultured and the number of people will work in the laboratory with their assigned tasks must be clearly defined. Besides that, maintenance of the stem cell lines and their characterization should be taken into account. If there is more than one cell lines are cultured simultaneously, the safeguards to prevent cross-contamination must be taken. The way new cell lines are being introduced, how are they going to be kept or quarantined (storage) for long use, the maintenance, the proper disposal of waste should be questioned and answered clearly. Basically the stem cell laboratory is not much different from the other cell culture laboratories despites some additional special techniques because of the uniqueness of the stem cells behavior (Wesselschmidt & Schwartz, 2013).

Physical Plan

Preplanning of laboratory design and layout first and foremost must be based on the budget. This is to ensure long term cost and continuous support in maintaining the stem cell cultures. (Wesselschmidt & Schwartz, 2013). The primary consideration in designing the cell culture laboratory is to provide the atmosphere that does not contaminate the cells and the people around. Thus, in the case of redesigning the laboratory, the location and its prior use must be checked to avoid any possible contaminations (Inamdar et al., 2012).

Laboratory Design

Lean principle can be introduced to reduce work travel around laboratory and cross-contamination (Griffiths et al., n.d.). The layout of laboratory as shown in Figure 1 is designed to accommodate six main essential requirements which are sterile handling, preparation, incubation, wash-up, sterilization and storage as described in Table 3. The preparation area supposed to be next to wash-up and sterilization areas meanwhile the sterile working area is accessible to the storage and incubators (Freshney, 1994).

The supply of liquid nitrogen and CO2 is needed in cell culture laboratory, thus their installation and handling must be given serious attention. The gases must be filtered from microbes, securely fixed, and its location must be far from cell culture work to avoid contamination during replacement. The full knowledge about equipment is necessary to ensure its correct function. The laboratory should have a reliable electric supply with the provision of uninterrupted power supply (UPS) for essential equipments in cell culture procedures such as class II cabinets, air filter and incubators (Inamdar et al., 2012).

Laboratory Operation

The core of stem cell laboratory operation is requirement for regular cleaning by its well-trained staff and strict monitoring procedures by a nominated member. Appropriate ventilation must be provided to avoid potential sources of dampness or fungal growth. As the lab do not have a “clean room”, it can use a high quality air conditioner and dehumidifiers together with hygienic practices such as the use of laminar flow hood, disposable items, clean gloved hands and disinfectant with 70% isopropanol in water (Inamdar et al., 2012).

Facilities or Equipments

Considerations

Sterile area

  • Clean, quiet and no through traffic.
  • Separate from animal house and microbiological laboratories.

Preparation area

  • Better to have prep room and culture area separated.

Wash-up area

  • Sink.
  • To ensure the hands are free from germs before and after the experiment.
  • Get access to the running water when in need e.g. burning, chemical spill.

Tissue culture area

  • Class II Biosafety Cabinet (BSC)
  • CO2 incubator.

Microscopy

  • Phase-contrast microscope.
  • Dissecting microscope.
  • Photo port.

Water bath

  • 37 ËšC constant temperature.

Centrifuge machine

  • Low speed centrifuge.

Vacuum source

  • Portable.
  • Supplied by the building.

2-L Erlenmeyer flasks with in-line filter

  • To collect aspirate.

Pipet

  • Automatic pipettor, rechargeable, Pasteur pipetor.

Micropipettors

  • 2, 20, 200 and 100 µl

Storage areas:

  • Liquids – ambient – 4ËšC – 20ËšC
  • Glassware.
  • Plastics.
  • Specialized equipment – cupboard, drawer.
  • Small items.
  • Chemicals – ambient – 4ËšC – 20ËšC (keep in sealed container).
  • Liquid N2 freezer.

Table 3. Essential requirements in tissue culture facilities. Modified from (Wesselschmidt & Schwartz, 2013 & Freshney,1994).

D:\sheffield stuff\stem cell report\stem cell lab layout 1.jpg

Figure 1. The layout of the stem cell laboratory with adjacent washing-up area, sterilization area, preparation area, administration, the storage and the culture area suitable for 20 to 30 persons. Image modified from (Freshney, 1994).

Embryonic Stem Cell Culture Analysis and Quality Control

Upon receiving the new stem cell lines, they must be quarantined until mycoplasma testing is done. Mycoplasma can spread rapidly between cultures and cause defect to cell lines (Young et al, 2010). Feeder cells also should be tested with this test and if the feeder cells are derived from mouse embryo, they must undergo microbiological test (Inamdar et al., 2012).

It is crucial to monitor hESC cultures as they can undergo genetic drift over time as they are being kept for a long time . Karyotyping must be done as soon as the cells are obtained to see the changes. Besides that, in order to ensure their pluripotency, several assays can be done routinely such as checking for antibody markers through immunofluorescent staining or quantitative PCR and observation of cell morphology. The gold standard to ensure pluripotency is through formation of teratoma tumor in immune-deficient mouse by transplanting a hESC cell line (Lyons et al., 2007).

Skilled Personnel

Staff must be competent to fulfill their duties effectively thus they must be equipped with necessary trainings such as in handling laboratory safety and equipments, aseptic techniques, cell banking, characterization of cell lines and their safety testing, quality control and record management (Inamdar et al., 2012). The knowledge of methods to keep the hESC in undifferentiated cells is crucial. For the time being automation is not necessary in stem cell culture as it requires eyes to passage the cells and to determine their confluent is subjective. Secondly, the protocol is developing and the researchers are improving with it thus there is no need for automation as the procedure might change in the following experiment (Koppal, 2013).

In conclusion, in order to operate a successful stem cell laboratory such considerations must be reviewed which includes documentation and approvals, established Standard Operating Procedures, laboratory planning and design, proper equipment and electric supplies, quality control and skilled personnel in order to ensure smooth operations and reproducible data.

REFERENCES

Freshney, IR. Laboratory Design and Layout. In: Ian Freshney, R., editor. Culture of Animal Cells: a manual of basic technique. 5. New Jersey: John Wiley & Sons; 1994. p. 19-23.

Griffiths, T., Peto, A., Thorogood, J., & Tiffany, D. (n.d.). Conceptual Design of a Cutting Edge Stem Cell Research Facility . Retrieved from http://www.engineering.leeds.ac.uk/e-engineering/documents/Griffiths_et_al_for_publication.pdf on 15th May 2014

Inamdar, M. S., Healy, L., Sinha, A., & Stacey, G. (2012). Global Solutions to the Challenges of Setting up and Managing a Stem Cell Laboratory, 830–843.

Lyons, I.; Tan, D.; Schwartz, PH.; Rao, M. Setting up a facility for human embryonic stem cell research. In: Loring, JF.; Wesselschmidt, RL.; Schwartz, PH., editors. Human Stem Cell Manual: A Laboratory Guide. 1. New York: Elsevier; 2007. p. 389-413.

Koppal, T. (2013, December 6). Ask the Expert: Setting up a Stem Cell Culture Lab: Dos and Don'ts. Lab Manager. Retrieved from http://www.labmanager.com/ask-the-expert/2013/12/ask-the-expert-setting-up-a-stem-cell-culture-lab-dos-and-don-ts#.U37w6nb93D8 on 20th February 2014

UK Stem Cell Tool Kit. Department of Health. Retrieved from http://www.sc-toolkit.ac.uk/regulatoryroutes.cfm on 20th May 2014

Wesselschmidt, R. L., & Schwartz, P. H. (2013). The Stem Cell Laboratory: Design, Equipment, and Oversight. NIH Public Access, (6), 3–13.

Young, L., Sung, J., Stacey, G., & Masters, J. R. (2010). Detection of mycoplasma in cell cultures. Nature Protocols, 5, 929–934.

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