APPENDIX

Laboratory Report Template

 FACULTY OF ENGINEERING TECHNOLOGY

DEPARTMENT OF CHEMICAL ENGINEERING TECHNOLOGY

(INDUSTRIAL BIOTECHNOLOGY)

SCHOOL OF BIOPROCESS ENGINEERING

LABORATORY REPORT
CODE & COURSE : PTT 305 CELL & TISSUE CULTURE
TECHNOLOGY
NO. OF EXPERIMENT : Experiment 2 CULTURING
ANCHORAGE-DEPENDENT ANIMAL CELL LINE
TITLE

Group: B1

Members (Name & Matric):

1. LOH SOON BOON 151282504

2. KEE HUI MIN 151282500
3. MUHAMMAD BADRUL AMIN BIN MUHAMMAD RAFI 151282511
4. NURUL HIDAYAH BINTI MAMAT 151282533
5. NURFAZIYATUL AIZA BINTI AMRAN 151282529
6. NUR ATHIRAH BINTI MOHD RADZI 151282523

Lecturer: SIR JOHAN ARIFF MOHTAR

Date of Experiment: 18/9/2017

OBJECTIVES:

1. To prepare the complete culture medium for animal cells.

2. To culture anchorage-dependent animals under aseptic condition.

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INTRODUCTION/BACKGROUND:

Primary cell culture is the maintenance of growth of cells dissociated from the parental
tissue (such as kidney or liver) using mechanical or enzymatic methods, in culture medium using
suitable glass or plastic containers. The primary cell culture could be of two types depending
upon the kind of cells in culture.

Adherent cells are cells shown to require attachment for growth are said to be anchorage
dependent cells. The adherent cells are usually derived from tissues of organs such as kidney
where they are immobile and embedded in connective tissue.

Suspension cells are cells which do not require attachment for growth or do not attach to
the surface of the culture vessels are anchorage independent cells or suspension cells. All
suspension cultures are derived from cells of the blood system because these cells are also
suspended in plasma in vitro for example, lymphocytes.

Secondary cell cultures is when a primary culture is sub-cultured, it becomes known as

secondary culture or cell line. Subculture (or passage) refers to the transfer of cells from one
culture vessel to another culture vessel. This is periodically required to provide fresh nutrients
and growing space for continuously growing cell lines. The process involves removing the
growth media and disassociating the adhered cells (usually enzymatically). Such cultures may
be called secondary cultures.

Cell Line: A cell line or cell strain may be finite or continuous depending upon whether it
has limited culture life span or it is immortal in culture.

Complete Culture Media in the early years, balanced salt solutions were supplemented with
various nutrients (amino acids, vitamins, serum etc.) to promote proliferation of cells in culture.
Eagle was a pioneer in media formulation. He determined (during 1950-60) the nutrient
requirements for mammalian cell cultures. Many developments in media preparation have
occurred since then. There are more than a dozen media now available for different types of
cultures such as EMEMEagles minimal essential medium, DMEMDulbeccos
modification of Eagles medium and CMEMGlasgows modification of Eagles medium.

Anchorage-dependent cells are of great interest for various biotechnological applications.

They represent a formidable production means of viruses for vaccination purposes at very large
scales (in 10006000 l reactors) using microcarriers, and in the last decade many more novel
viral vaccines have been developed using this production technology. With the advent of stem
cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes,
the development of novel culture devices and technologies for adherent cells has accelerated
greatly with a view to the large-scale expansion of these cells.

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METHODOLOGY:

Preparation of Complete Culture Medium

1. The specification of medium was checked and the conditions that are required was
determined.
2. The medium and other supplement or addition that required was placed into biosafety
cabinet.
3. Any outer wrapping was removed and the medium bottle was swabbed with 70% ethanol.
4. The bottle was uncapped.
5. 10% of FBS (3ml) was added to the DMEM medium.
6. 5% of (1.5ml) Pen/Strep and 2mm L-glutamine was added and the bottle was recapped.
7. The bottle was labelled with data and initials and used directly.

Thawing and Culturing Human Breast Adenocarcinoma (MCF-7) Cells

1. The vial was thawed by gentle agitation in a 37C water bath. The cap was kept out of
the water to reduce the possibility of contamination.
2. After thawing, the vial was swabbed thoroughly with 70% ethanol and was handled in a
biosafety cabinet.
3. The vial contents was transfer to a 15ml Falcam Tube in which contained 5ml complete
culture medium.
4. The suspension was centrifuge for 5minutes at 1000rpm. The supernatant that contained
the cryoptrotectant was discarded.
5. The cell pellet was resuspended in the 6ml complete culture medium.
6. The cell suspension was dispensed into 6-well culture plate (3 replicates). Another 2ml
complete medium was added into each well.
7. The plate was shaked firmly to evenly scatter the cells.
8. The plate was incubated at 37C in CO2 incubator.
9. The cell culture was observed under the inverted microscope after 24 hours. Digital
eyepiece was used if required.

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RESULTS:

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DISCUSSION:

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CONCLUSION:

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REFERENCES:

1. University of Kent. Moodle External. Study Guide - Biotechnology and Cell Culture.
Retrieved 18 Sep 2017 from
https://moodle.kent.ac.uk/external/mod/book/view.php?id=2604&chapterid=160
2. Nandkishor Jha . 2017. Culture Media for Animal Cells: An Overview. Retrieved 18
Sep 2017 from http://www.biologydiscussion.com/biotechnology/animal-
biotechnology/culture-media-for-animal-cells-an-overview/10499
3. Otto-Wilhelm Merten. 2015 Feb 5. Advances in cell culture: anchorage dependence.
Retrieved 18 Sep 2017 from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275909/

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QUESTIONS & ANSWERS:

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