UNIVERSITI TUNKU ABDUL RAHMAN FACULTY OF ENGINEERING AND SCIENCE

UEMB 4193 Tissue Engineering Lab 1 Maintenance of Anchorage Cells

NAME ID COURSE DATE OF EXPERIMENT

: : : :

Soong Wen Ying 1101380 Biomedical Engineering 17th FEBRUARY 2014

Introduction: Maintaining cells in tissue culture is a fundamental and vital procedure in tissue engineering. Maintaining the cells culture enables the rate of cells growth and proliferation to increase. Thus, through this experiment, the fundamental skills of maintaining the cells culture are able to be practiced and learnt. Besides, the knowledge about primary culture and cell line after subcultured is being understood and studied.

Methods and materials The whole procedure was performed in aseptic environment to prevent contamination. Thus the whole experiment was done in laminar flow hood. The vertical laminar flow hood was chosen to be used in our molecular biology lab. The laminar flow hood is a high level of product protection to protect the work area from dust and bacteria contamination. It is an enclosed bench which has continuous displacement of air that passes through a HEPA (high efficiency particle) filter. The HEPA filter is a type of air filter removed 99.97% of all particles greater than 0.3 micrometer from the air pass through. In the vertical laminar flow the HEPA filer flows down from the top of the cabinet. The vertical laminar flow hoods will protect the user and the cell culture. And since it is with vertical flow, we need to avoid overcrossing on the containers and all of them are placed in different horizontal line to prevent cross contamination. Besides, the vertical laminar flow hood is fit out with short wave UV light which is use to sterilize the surfaces of the work area and tools in the enclosed bench. The UV light should be turn on for 30 minutes before being used. However, the UV light can harmful human skin and eye so we must stay away the vertical laminar flow to avoid damage when the UV light is on. Apart from that, there are some tools are place inside the vertical laminar hoods such as Bunsen burner and pipette. The function of the Bunsen burner is to be used for heating and sterilization. Every time we open and close container of cells and medium, we need to flame them to avoid contamination. The pipette is an important device to measure the volume of fluid near the true value. To avoid contamination, we are not allowed to use same pipette tip for different fluid. Besides that, we also need to prepare 70% ethanol to spray on the inner and outer surface of the vertical laminar flow hood and CO2 incubator and wiping clean. Furthermore, the carbon dioxide (CO2) incubator is a device to grow and maintain the cell cultures. The CO2 incubator maintains 5-10% carbon dioxide in atmosphere inside which is suitable environment to let the cell grow. Besides, it also maintains the optimal temperature which is 37oC, humidity and pH value in the atmosphere inside.

Procedure: 1. 2. 3. 4. 5. 6. 7. The medium was thawed in 37C water bath. 9ml of fresh medium was transferred into 25cm3 T-flask. The flask was conditioned at 37C CO2 incubator for 15 minutes. HeLa cell was thawed by rubbing in hand. 1ml of HeLa cell was transferred into T-flask. Cell was observed under microscope. Cell was incubated in CO2 incubator.

Procedure of subculturing HeLa 1. 2. 3. 4. 5. 6. 7. 8. 9. The old medium was removed. Cell was rinsed in 3ml PBS. PBS was removed. 2ml of trypsin (0.25%)-EDTA was added. The cell was incubated for 2-3 minutes and cell detachment was checked under microscope. 8ml fresh medium was added into flask. It was centrifuged with speed of 1000rpm for 5 minutes. Medium was discarded and 10ml of fresh medium was added. It was dispensed into new flask.

Discussion Cell culture is the technique of growing and maintaining the cells of multicellular organisms within an artificially controlled environment to stimulate growth of the cells. There are two different ways to culture cells. These two ways are adherent cultures and suspension cultures. Both are used in culturing adherent and suspension cells respectively. Hence, there are differences and difficulties in culturing adherent and suspension cells. Generally, adherent culture is an anchorage-dependent culture while suspension culture is an anchorage-independent culture. Adherent culture enables the cells to grow attached to the surface of a flask while suspension culture consists of rounded cells floating in medium or lightly adhered to the flask. Adherent cultures are used for cells which required to be attached to a surface while suspension cultures are used for cells which naturally exist without adhering to any surfaces. Both types of cell culture need growth media or medium. However, the growth media or medium in suspension cultures are most likely to be in liquid form. On the other hand, the growth media or medium in adherent cultures would be solid to allow the cells to attach and proliferate. Such being a case, adherent cells culture would normally requires tissue-culture treated vessel to enable the cells adhering the vessel to grow. For suspension cells culture, it

could be maintained in culture vessels that are not tissue-culture treated but it requires agitation such as shaking or stirring for adequate gas exchange. Next, the difference in culturing adherent and suspension cells is the way of the cells passaging. Adherent cells culture requires periodic passaging as the cells only grow on a monolayer. However, this monolayer and periodic passaging allows easy visual inspection under the inverted microscope. Meanwhile, the suspension cells are easier to passage during culturing as they can dilute culture to stimulate growth. For suspension cells culture, it requires daily cell counts and viability determination to follow growth patterns of the cells culture. This might lead to the time spending in observing extended and increased compares to observation of adhesion cells culture. For adherent cells culture, the adherent cells need to be dissociated enzymatically or mechanically as the characteristic of adherent cells caused the cells are strongly attached to the surface of the medium during feeding and harvesting process of the cells culture. Thus, enzyme is required to loosen the cells from the wall or surface and transfer to falcon tube. The enzyme which used to dissociate the adherent cells and remove the cells from the surface of medium is trypsin. Another method to dissociate the adherent cells would be by spraying the surface by using pipette techniques mechanically. This method needs to be handled carefully to avoid the damage of the cells. On the other hand, suspension cells culture does not require any enzymatic or mechanical dissociation as the suspension cells are flow on the medium. The separation of the medium and cells could be done easily by centrifugation. Another difference and difficulties in culturing adherent and suspension cells is the ability to observe the cells. Adherent cells culture which is only a monolayer allows easy visualization of the cells while the suspension cells culture are floating around with the liquid medium is harder to view and observe. The suspension cells culture are floating with the liquid medium and consists of many layers of cells. This has lead to the difficulty to observe through the microscope as the enlargement of the microscope images need to be careful to avoid inaccuracies. Lastly, the growth of the adherent cells culture is limited by surface area as the cells could only attach and grow on the surface area of the vessel or medium provided. As a consequence, the product yields in adherent cells culture may be limited. For suspension cells culture, the growth is limited by the concentration of cells in the medium as the cells are fed up by the medium. This would allow the ease of scaling-up for the suspension cells culture. There are also some limitations and challenges in cells culturing. In cell culture, there would be some missing features for the cells that have grown. These missing features are the original blood circulation, original tissue organization and structure, factors in blood, hormones, and confluency and contact with other cells. The challenges to grow and harvest cells culture are mostly caused by the total surface area and the density of the cells at harvest. Sometimes, the

cells might not grow in the way which we wanted and also infected by contaminants. Thus, observation should be done frequently and sterilization should be done and ensured during cells culturing.

References 1. Adherent Cell Culture vs. Suspension Cell Culture (2013). Retrieved from http://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics/celllines/adherent-vs-suspension-culture.html 2. Group 4 Project Cell Culture (2010). Retrieved from http://php.med.unsw.edu.au/cellbiology/index.php?title=Group%E2%80%834%E2%80% 83Project%E2%80%83-%E2%80%83Cell%E2%80%83Culture 3. Jon Rowley, Eytan Abraham, Andrew Campbell, Harvey Brandwein, Steve Oh. (2012). Meeting Lot-Size Challenges o Manufacturing Adherent Cells for Therapy. Retrieved from http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_WhitePapers_Meeting_ Lot-Size_Challenges_of_Manufacturing_Adherent_Cells_for_Therapy.pdf 4. Cell Culture. (2008). Retrieved from http://www.molecularstation.com/wiki/Cell_culture#Limitations_of_Cell_Culture