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Laboratory Design and Layout

ATC : Lecture 3
Laboratory Design, Layout and Equipment Aseptic Technique Contamination

- major requirement that distinguishes tissue culture from most other laboratory techniques is the _______________________ - Important that tissue culture laboratory be __________ and have __________________.

Requirement for designing a tissue culture laboratory

number of users - How many people will work in the facility? working duration per week? What kinds of culture will they perform? space Culture operation : wash up,preparation,sterilization : storage : incubation containment and sterility - Ensure the air pressure balance between biohazard protection and maximum sterility

location of preparation area - Can be located either close to the aseptic area or in separate room storage - Place to store disposable plastic wares and liquid nitrogen tank Access - Provide space for access for maintenance and installation of equipment

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Tissue Culture Laboratory Facilities

Main Functions 1) Sterile handling area: clean and quiet, no through traffic, separate from animal house and microbiological labs 2) Preparation area 3) Wash-up area 4) Incubation area

5) Storage areas (a) Liquids : RT, 4,-20,-80C (b) shelf (glassware and plastics) (c) drawers (small items ) (d) Chemicals : RT, 4,-20C (share with liquids but keep chemicals in sealed container over desiccant) 6) Sterilization

Essential Equipment
1) Laminar-flow Hood (class II biological safety cabinets)

Horizontal laminar flow

- Airflow blows from the side facing operator, parallel to the work surface - Give the stable airflow and best sterile protection to the culture and reagents

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Vertical laminar flow

2) Incubator 3) Sterilizer 4) Centrifuges 5) Refrigerators and freezers 6) Inverted microscope 7) Water purification 8) Cryostorage container 9) Balance 10) Hemocytometer

- Air blows down from the top of the hood onto the work surface and drawn through the work surface -

Aseptic technique
- Any technique incorporating measures that prevent the contamination of cultures, sterile media,etc and/or the inappropriate infection of persons, animals or plant, by extraneous microorganisms. - Aims to exclude contamination by establishing a strict code of practice and ensuring that everyone using the facility adheres to it.

Element of aseptic environment 1) Quiet area 2) Work surface 3) Personal hygiene 4) Reagents and media 5) Cultures

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Contamination
- the presence of any contaminant (bacteria, yeast, mycoplasma) and etc; - the presence of harmful or invasive microorganisms in a population of desirable ones. Sources of contamination 1) Operator technique 2) Environment 3) Use and maintenance of laminar flow hood 4) Incubators 5) Storage vessels

a) bacteria

b) yeast

c) molds

d) fungi

Types of microbial contamination

http://zt.invitrogen.com/site/tw/zt/home/References/gibco-cell-culture-basics/biological-contamination/yeastcontamination.html

e) mycoplasma

http://www.disease-picture.com/mycoplasma-infection-cell-culture/ http://www.molecularstation.com/forum/cell-biology-cell-culture/1888-cell-culture-fungal-contamination.html

Monitoring for contamination 1) Examine the cells visually and with a microscope before each operation a) A sudden change in pH pH = pH = b) Cloudiness in the medium

c) Presence of particles that different from the cells - Bacteria shimmering granular/rods - Yeast - round/ovoid particles - Fungi thin filamentous mycelia 2) Record the nature of contamination when one occurs 3) When working with different cell lines in parallel, pay close attention to avoid cross-contamination

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Mycoplasma
- The smallest and simplest self-replicating organisms - They cannot perform many metabolic functions such as cell wall production or synthesis of nucleotides and amino acids - Are strictly parasites - Parasitize a wide range of organisms including humans, animals, insects and plants - Incidence of mycoplasma contamination 1% of primary cell cultures 5% of early passage cell cultures 15-35% of continuous human or animal cell lines

Routes to Contamination
- Most common contamination mycoplasma species Natural host Human Species (frequency) M.Orale (20-40%), M.fermentas (10-20%) , M.hominis (10-20%) M.Arginini (20-30%), A.laidlawii (5-20%) M.Hyorhinis (10-40%) 1) Technique Manipulations, pipetting, dispensing etc touching or holding pipettes too low down, touching necks of bottles, inside screw caps 2) Laboratory staff Dust from skin, hair, or clothing dropped or blown into the culture Aerosols from talking, coughing, sneezing, etc

Bovine Swine

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3) Materials and reagents Inadequate sterilization procedures poor commercial supplier 4) Equipment and instruments laminar-flow hood : filter needs to change periodically CO2 incubator : growth of mold in a humid atmosphere pipettes, bottles and etc : ineffective sterilization 5) Incoming cell lines contaminated at the source or during transit

Effect of mycoplasma contamination on cell cultures a) General effects on eukaryotic cells : - Altered levels of protein, RNA and DNA synthesis - Alteration of cellular metabolism - Induction of chromosomal aberrations - Alteration of cellular morphology - Induction/suppression of cytokine expression - increase/decrease of virus propagation

Routine detection of mycoplasma contamination

b) Specific effect on hybridomas :
CELL LINE : NEW ARRIVAL

- Inhibition of cell fusion - Influence on selection of fusion products - Interference in screening of monoclonal antibody reactivity - Monoclonal antibody against mycoplasma instead of target antigen - Reduced yield of monoclonal antibody
Quarantine culture laboratory Initial Mycoplasma testing PCR Fluorescence staining (DAPI/Hoechst)

Elimination

Mycoplasma positive

Mycoplasma negative

Sterile culture laboratory

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Detection of mycoplasma contamination

a) - Cultures are stained with fluorescent dye (Hoechst 33258) - DNA fluoresces brightly when examined under a UV microscope - widely used and recommended indirect test- easy and relatively fast

Fluorescence Detection of Mycoplasma

http://www.shigen.nig.ac.jp/shigen/news/n_letter/2008/newsletter_v4_n6En.html

Protocol : fluorescence detection of mycoplasma 1) Seed indicator cells (3T6/NKR/A549) into Petri dishes w/o antibiotic 2) Add 1.5 ml of medium from the test culture

6) Fix cells with acetic methanol for 10 min 7) Wash cells with deionized water 8) Add Hoechst 33258 and stain for 10 min at RT 9) Discard and rinse the stain with water

3) Incubate the culture until the cells reach 50-60% confluence 4) Remove the medium and discard it. 5) Rinse cells with free-phenol red medium

10) Examine by epifluorescence check for extranuclear fluorescence (cytoplasm and intercellular spaces)

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Protocol : PCR detection of mycoplasma

b) - ______________________ are used as target sequences - Sensitive and specific assay low expenditure of labor, time and cost, reproducibility and documentation of result.

A) Sample collection and preparation of DNA 1) Culture the cell line to be tested w/o antibiotic for several days 2) Collect 1 ml of the supernatant medium 3) Centrifuge for 13,000 g, 5 min 4) Resuspend in 1 ml PBS buffer by vortexing 5) Repeat step 3) and resuspend the cell pellet in PBS buffer by vortexing and heat to 95C,15 min 6) Extract and purify the DNA

B) PCR Reaction i) Set up 2 reactions per sample to be tested sample only : dNTPs, Myo-5, Myco-3, 10xPCR buffer, dH2O sample and DNA internal standard : dNTPs, Myo-5, Myco-3, 10xPCR buffer, dH2O, internal control DNA Water control (negative control) : dNTPs, Myo-5, Myco-3, 10xPCR buffer, dH2O iv) Electrophorese the PCR product on agarose ii) Add Taq DNA polymerase and DNA sample/positive control DNA iii) Run the PCR

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Mycoplasma detection by PCR

Sample result

Alternative methods for detecting mycoplasma 1) - Arginine deiminase/nucleoside phosphorylase - detect mycoplasma with specific enzymes - 6-methylpurine deoxyriboside (Mycotest, invitrogen) detect toxicity 2) Molecular hybridization

-ve

+ve +ve & -ve treated

+ve

error +ve

- Conventional molecular hybridization techniques - Southern blot analysis

Uphoff CC and Drexler HG (2002) Mycoplasma contamination of cell cultures : Incidence,sources,effects,detection,elimination,prevention.Cytotechnology 39:75-90

2) Microbiological culture - Cultured cells are seeded into mycoplasma broth, grown for 6 days and plated out onto special nutrient agar- check for the colonies - Very sensitive method but much slower and more difficult to perform - Mycotrim - Commercial kits for microbiological detection 4) Immunological assays - Use mycoplasma-specific polyclonal/monoclonal antibodies to detect - Applied in immunofluoresence and enzymelinked immunosorbent assays (ELISA)

Elimination methods of mycoplasma contamination 1)Physical procedures : - heat treatment - filtration through microfilters 2) Chemical procedures : - exposure to detergents - washings with ether-chloroform - treatment with methyl glycine buffer 3) Chemotherapeutic procedures : - antibiotic treatment in standard culture (BM-cyclin, ciprofloxacin, tylosin and etc) - soft agar cultivation with antibiotics

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Prevention of mycoplasma contamination

a) Cell culture facility Certified laminar-flow should be used and regularly checked Incubators should be regularly cleaned and controlled Cell culture materials should be properly disposed off central sterilization

b) Cell culture procedures Cell cultures/supplement/media should be purchased from reputable supplier Incoming cell cultures should be kept in quarantine until proven sterile Mycoplasma testing should be performed regular intervals Mycoplasma-positive cultures should be immediately discarded and treated

c) Operator technique Handling of only one cell line at a given time. Different medium aliquots for different cell lines No pouring, but pipetting of medium from bottles or flasks Prohibition of mouth pipetting Written laboratory records for every cell culture

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