Cell Culture Module BME 306 © Devina Jaiswal Biosafety Hood • Working environment is protected from dust and contamination by a constant, stable flow of filtered air passing over the work surface. • It provides a sterile environment for cell culture. © Devina Jaiswal Incubator Maintains ambient culture conditions • 5-6 % CO2 • 37°C temperature © Devina Jaiswal 5-6 % CO2 : • Most mammalian cells are cultured at pH 7.4 • Growth medium contains buffering agents • including an organic (e.g., HEPES) or CO2-bicarbonate based buffer • Changes in the atmospheric CO2 can alter the pH of the medium. • That is why exogenous CO2 used in an incubator 37°C temperature What happens if cells are cultured outside incubator? • How would you maintain the ambient conditions? • What are the alternatives for CO2 supply? © Devina Jaiswal What happens if cells are cultured outside incubator? • They will not survive as room temperature is not ambient for their survival. • pH of the media would change. How would you maintain the ambient conditions? • One can add a heated stage or additional warmer to maintain 37 °C temperature. • HEPES buffer can be added to media to compensate for CO2 supply. • Make sure the area with cells is not contaminated What are the alternatives for CO2 supply? • HEPES buffer © Devina Jaiswal Flask Cell culture dishes • Made up of polystyrene, disposable • Available cell culture dishes: Cell Treat, Corning • Flasks: 75, 175 mm • Petri dishes: 150, 100, 60, 35 mm • Cell culture treated dishes • Untreated dishes © Devina Jaiswal Filtered cap Cell culture surface Petri Dish Pipets and Pipet Aids 1. ___Red band:25 ml______ 2.___Orange band: 10 ml___ 3. ___Blue band: 5 ml_____ 2 1 P 1000: Blue : max 1000 µl P 200: Yellow : max 200 µl P 20: Yellow : max 20 µl P2: Red : max 2 µl 3 © Devina Jaiswal Centrifuge • Cell suspension requires centrifugation: • increase the concentration of cells • wash off a reagent • Cells sediment satisfactorily at 80-100g. • Difference between RPM and RCF • RPM = rotations per minute • RCF = relative centrifugal force (# of g) • g = 1.12 x10-5 * radius (cm) * RPM © Devina Jaiswal Media • Supplemented with: • • • • essential amino acids salts vitamins glucose • pH 7.4 • some normal fibroblast lines perform best at pH 7.4 - 7.7 • Phenol red: indicator of change in pH. © Devina Jaiswal Serum • Supplies needed growth factors and nutrients • Standard serum for tissue culture cells is calf serum • Some cells like horse serum • Some cells require the fetal calf (also known as fetal bovine) serum (FBS) • More expensive - $ • Some cells (usually human) require serum of their own species • Most cells require 5-20% serum for good growth • We will use 10% FBS. © Devina Jaiswal Cells Cell lines: • Immortalized cells • Cell-line cells are adapted to culture plate environment • Often differ genetically and phenotypically from their tissue origin Primary Cells: • isolated directly from living tissue • Have finite expansion capacity • Retain their morphology and markers © Devina Jaiswal Sterilizer • High temperature “steamer” and “pressure cooker” • generates higher than 100kPa • Liquids • some can be autoclaved • others with heat sensitive proteins should be filter sterilized • Glassware • autoclaved • Trash • autoclaved • Temperature sensitive items • cleaned with ethanol • In general, UV light is not a good sterilizer, but can be used in conjunction with ethanol. © Devina Jaiswal Trypsinization • Cells should be passaged when they are 70-90% confluent • What is confluency: • The percentage of surface area of culture dish covered by adherent cells. • What is Trypsin? • It is an enzyme that cleaves proteins. • It is found in digestive system of many vertebrates. © Devina Jaiswal Cell counting using hemocytometer © Devina Jaiswal 𝑵𝒖𝒎𝒃𝒆𝒓 𝒐𝒇 𝒄𝒆𝒍𝒍 𝒔Τ𝒎 𝒍 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝒇𝒂𝒄𝒕𝒐𝒓 = 𝑻𝒐𝒕𝒂𝒍 # 𝒐𝒇 𝒄𝒆𝒍𝒍𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 × × 𝟏𝟎, 𝟎𝟎𝟎 𝒄𝒆𝒍𝒍 𝒔Τ𝒎 𝒍 # 𝒐𝒇 𝒔𝒒𝒖𝒂𝒓𝒆𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 © Devina Jaiswal © Devina Jaiswal 1     Phase 1: Hemostasis Phase Phase 2: Defensive/Inflammatory Phase Phase 3: Proliferative Phase Phase 4: Maturation Phase ◦ Protein synthesis & wound contraction ◦ Remodeling © Devina Jaiswal 2  Primary Contributors to Hemostasis ◦ Vasoconstriction ◦ Platelet aggregation ◦ Fibrin deposition 3  Primary Contributors to Hemostasis ◦ Vasoconstriction ◦ Platelet aggregation ◦ Fibrin deposition  Platelets release: ◦ Growth factors ◦ Fuel compounds ◦ Cytokines  End product of this stage is clot formation 4  Primary purpose: ◦ Bring inflammatory cells to injured area – leukocytes, macrophages ◦ Eliminate debris from dying cells  Characterized by: ◦ Erythema – skin reddening  Increased vascular permeability  Vasodilation occurs about 10 – 15 minutes after injury ◦ Edema – plasma leaks from intravascular space to extravascular space ◦ Heat ◦ Pain – fluid movement into injured area contributes to sensation 5   Debris clean-up Release nitric oxide ◦ has antimicrobial function   Release cytokines Stimulation of: ◦ Angiogenesis ◦ Fibroblast migration ◦ Collagen production  Forms scar  T-lymphocytes ◦ Lyse foreign cells 6   Various factors influence inflammatory cell apoptosis Macrophages remain in wound for approx. 7 days, then gradually slough off 7    About 7 days post-injury More cytokine release Fibroblasts & endothelial cells predominate ◦ Integrins expressed by leukocytes signal fibroblasts to migrate to wound area ◦ Fibroblasts secrete enzymes so they can pass through debris 8  Fibroblast: Present in the dermis layer of the skin. They are primarily responsible for generating the connective tissue and allowing skin to recover from injury. Day 3 Day 7 Day 5 © Devina Jaiswal 9  Key signal in wound healing is tissue hypoxia: ◦ Proliferating cells consume oxygen 3 – 5 times faster than cells in resting phases ◦ Signals cytokine release ◦ Stimulates angiogenesis  Endothelial sprouts derive from intact capillaries  One sprout reaches another sprout and they interconnect to form a new capillary. 10   Cell movement Cell movement is a highly dynamic process The process can be divided into 3 components 1. Protrusion of the leading edge 2. Adhesion of the leading edge and deadhesion of the cell body and rear 3. Cytoskeleton contraction to pull the cell forward © Devina Jaiswal 11     https://www.youtube.com/watch?v=VVgXDW_8O4U Actin is one of the most abundant and highly conserved proteins in eukaryotic cells. The globular protein (G-actin) assembles into long filaments (F-actin) forming different networks within the cytoskeleton. Actin filaments grow and shrink by attachment and detachment of G-actin monomers at the two filament ends. © Devina Jaiswal 12   Protein Synthesis Begins ~5 days after wounding ◦ Fibroblasts synthesize collagen  Collagen is main protein in scar tissue  Synthesis continues for 2 – 4 weeks, then tapers off ◦ Fibroblasts synthesize proteoglycans  Proteoglycans help fibril formation  Proteoglycans make skin viscoelastic 13     Wound contraction Begins ~4-5 days after wounding Continues for about 2 weeks Rate of contraction ◦ 0.6 – 0.7 mm/day ◦ Square wounds contract more quickly than circular wounds  Myofibroblasts are the main cell responsible for wound contraction 14     Remodeling Begins ~21days after wounding Continues for up to 12 months Process associated with wound breaking strength compared to normal dermis ◦ 1 week: 3% strength of normal ◦ 3 weeks: 20% strength of normal ◦ 3 months: 80% strength of normal 15 16 (A) scratch assay (B) stamp assay (C) thermal wounding (D) electrical wounding (E) optical wounding using laser © Devina Jaiswal 17 © Devina Jaiswal 18 1) 2) 3) 4) 5) 6) 7) 8) Take a confluent plate of fibroblast cells. Use a scraper to scratch the monolayer along the diameter of the petri dish. Using the 5x objective lens and ImageJ, determine the pixel size by capturing an image of a ruler & setting the scale. Take an image of the wound at 0hr, 24 hrs, 48 hrs, 7 days With each image, use ImageJ to measure the width of the wound at three positions along the scratch in your image. Use scale to convert the measured values from pixels to ‘mm’ Use ‘line’ tool to make a line across the wound and press ‘ctrl-M’. Create a bar graph to depict your results in the report. © Devina Jaiswal 19    Groups 1 & 3: Cell culture dish with normal media (control) Groups 2 & 4: Cell culture dish with actin polymerization inhibitor (Cytochalasin D) (Treatment) Both the groups will analyze their data separately. At the end of the study, the data will be shared. 6 Wound Width (mm)  5 4 3 2 1 0 0hr 24hr Control 48 hr CD Treated 7 days © Devina Jaiswal20           The report includes cell culture, cell counting and wound healing The report should include Title page, Objective (10 pts), Materials & Methods (20 pts), Results (40 pts), Discussion (25 pts) and Conclusion (5 pts) Objective: Have a clear objective that describes the goals of both the cell counting & wound healing studies. Materials and Methods: includes all the materials that you have used along with the methods followed during both the modules. This section should be in your own words. Results: include figures and cell counting results in results and discussion section Discussion: includes details about why certain trends are seen in a plot. Discuss if there is an anomaly and try to find the reason. Conclusion: It should not contain any new idea or details. It should be a summary of what was completed in the study. Make sure all the graphs have x-axis and y-axis labels, error bars (st. dev.), legend, title, and caption underneath. Figure caption should be brief. (1-2 sentences) Use 12pt, double space, Times New Roman © Devina Jaiswal 21 Day 0 wound helaing Day 1 Day 2 Day 7 Cell culture fig As you prepare your Wound Healing lab report this weekend, here are a couple of considerations. The live control sample had a known concentration of ~600,000 cells/mL. Each group had an unknown sample that should have had a similar count because the initial cell seeding density was the same as the control. The dead control sample's cell count can be very variable because some dead cells may burst open due to the ethanol, and then they show up as debris in the hemocytometer. Or because the cells sat out on the lab bench for a good while & weren't incubated, some could have died there, too. Remember, there is a scaling up factor of 10,000 cells/mL, so a small difference in your count can result in a large difference in the cell density calculation. There could have been some "injury" caused to the cells by a pipette error, scraping the side when you intended to go straight into the middle of the well. If you created a lot of bubbles in your sample, you may not have extracted a clear sample when you meant to... so think about some of your techniques that could cause your cell counts - alive & dead - to be different than what was expected. My prof said when she made the two cell culture samples - dead & alive - the cell counts were both 600,000 cells/mL. You'll want to use this cell count as a comparison when you compute the cell counts using the formula in your handout on page 7. The dilution factor in all three test samples was 2, because you mixed 20 uL of trypan blue with 20 uL of cell solution in each well, so you had twice the fluid content as the original cell solution. If any of your cell counts is very different from the 600,000 figure, then you'll want to explain the possible sources of error in your cell culture/wound healing lab report. BME 306 Spring 2019 Cell Culture Module Wet Laboratory Equipment Personal protective equipment: Gloves, closed toe shoes, lab coats Biosafety Hood The major advantage of working in a laminar flow hood is that the working environment is protected from _________ and _______________________ by a constant, stable flow of filtered air passing over the work surface. It provides a ____________ environment for cell culture. 1 Incubator: Maintains ambient culture conditions 5-6 % CO2 : Most mammalian cells are cultured at _______ °C Growth medium contains buffering agents including an organic (e.g., HEPES) or CO2-bicarbonate based buffer. Changes in the atmospheric CO2 can alter the pH of the medium. That is why _________ is used in an incubator. 2 37°C temperature What happens if cells are cultured outside incubator? How would you maintain the ambient conditions? What are the alternatives for CO2 supply? Flask Cell culture dishes: Made up of __________________ - they are disposable Available cell culture dishes: Cell Treat, Corning Flasks: 75, 175 mm Filtered cap Petri dishes: 150, 100, 60, 35 mm Cell culture treated dishes:_______________________ Untreated dishes:______________________________ 3 Cell culture surface Petri Dish Pipets, Pipettors and Pipet Aids Pipetman and serological pipets are used for larger volumes 1. _______________________ 2._______________________ 3. _______________________ 2 1 3 Pipet and pipet tip boxes are color-coded based on the volume of liquid. P 1000: Blue _____________________________________ P 200: Yellow _____________________________________ P 20: Yellow _____________________________________ P2: Red _____________________________________ 4 Centrifuge Cell suspension requires centrifugation to _______________________________ of cells or to _______________ off a reagent. Cells sediment satisfactorily at 80 - 100g. It is very important to remember to always balance a centrifuge. That means putting in a vial of the same volume directly across from your sample vial. Make sure that all removable parts are balanced, that all shields are matched, & that all buckets are the same, Otherwise the motor will be out of balance, the centrifuge will vibrate VIOLENTLY and none of this is good for the centrifuge. Difference between RPM and RCF RPM = rotations per minute RCF: relative centrifugal force (# of g) g = 1.12 x10-5 * radius (cm) * RPM Media Culture media is supplemented with essential amino acids, salts, vitamins and glucose. Initial attempts to culture cells were performed in natural media based on tissue extracts and body fluids, such as chick embryo extract, serum, lymph, etc. With the propagation of cells, consistent quality led to the introduction of chemically-defined media based on analyses of blood fluids and nutritional biochemistry. Most labs buy the medium already prepared and bottled or as a powder that must be re-hydrated and filter sterilized. Physiochemical Properties: pH - Most cell lines grow well at pH 7.4, although some normal fibroblast lines perform best at pH 7.4-7.7. Phenol red is used as an indicator of change in pH. 5 Serum Serum supplies needed growth factors and nutrients. Some cells like horse serum. The standard for tissue culture cells is calf serum. Some cells require the more expensive fetal calf (also known as fetal bovine) serum (FBS), and some cells (usually human) require serum of their own species. Most cells require 5-20% serum for good growth. In our study, we will use 10% FBS. Cells Cell lines: • • • Immortalized cells Cells are adapted to culture plate environment Often differ genetically and phenotypically from their tissue origin Primary Cells: • • • Primary cells are isolated directly from living tissue. The cells have finite expansion capacity The cells retain their morphology and markers Sterilizer A high temperature “steamer” and “pressure cooker”, which generates higher than 100kPa. The chamber should be evacuated after sterilization, to remove steam and promote subsequent drying; otherwise the articles will emerge wet, leaving a trace of contamination from the condensate on drying. Different ways to sterilize There are several ways you can sterilize instruments, liquids etc. While there are several ways to sterilize, some methods are usually better than others. Liquids – some can be autoclaved, others with heat sensitive proteins should be filter sterilized. Glassware – autoclaved Trash – autoclaved Temperature-sensitive items – sterilize with ethanol In general, UV light is not a good sterilizer, but can be used in conjunction with ethanol. 6 Cell Culture Protocol Thawing cells 1. When removing a vial from liquid nitrogen, twist the cap a little to release pressure in the vial (sometimes, the top pops open due to increase in internal pressure) 2. Prepare 4 ml medium (Fibroblast media+10% FBS + 1% Penn/Strep) and warm the media to 37 °C. 3. Freeze slowly, thaw quickly. 4. Defrost the vial of cells in a water bath. Let a small chunk of ice remain in the vial – this maintains temperature of the cells and thus prevents DMSO in the freezing medium from permeabilizing the cell membrane. 5. Prepare a 50 ml tube and add 20 ml warm media to it. 6. Pipette cells into warmed medium and centrifuge the tube at 1200 rpm for 5 mins. Decant media in the waste container and resuspend the cells in 10 ml warm media. 7. Transfer the cell suspension in the 100 mm dish and label with date and passage number. Place the dish in the incubator. This protocol is for 60 mm dish. Please adjust the volumes depending on the volume of the petri dish or culture flask being used. Trypsinization (Cells should be passaged when they are 70-90% confluent) What is confluency: ______________________________________________________________________________ ______________________________________________________________________________ 1. Warm the media sterile PBS (phosphate buffered solution) and Trypsin to 37 °C. What is Trypsin? 2. Remove the plate from the incubator. Remove the media from the plate and drain it in the waste container. 3. Wash the plate with 1 ml of PBS twice. Why do we wash the plate with PBS? ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ 4. Add 0.3 ml trypsin to the plate and place the plate in the incubator for two minutes. Monitor the plate and look at the cells under the microscope. 5. Add 5 ml media to 15 ml tube. Once 80% cells have detached, transfer the cells/ trypsin suspension to the 15 ml tube. 6. Centrifuge the tube at 1200 rpm for 5 mins. Decant the supernatant and resuspend the cells with 1 ml fresh media. 7. Use the cell suspension to count the cells. 7 Recommended Volumes Dish Style 100 mm 60 mm 1 well 2 wells 4 wells 8 wells Surface Recommended Area (cm2) Volume 55 13 21 5 9.4 3 4.2 1.5 each 1.8 0.7 each 0.8 0.3 each Volume Trypsin 1 0.3 Cell counting using hemocytometer The cells are placed in a gridded slide called a hemocytometer, and are counted manually under a microscope. 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠/𝑚𝑙 = 𝑇𝑜𝑡𝑎𝑙 # 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 8 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 × 10,000 𝑐𝑒𝑙𝑙𝑠/𝑚𝑙 # 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 Lab Activity 1 – February 4, 2019 Cell culture demonstration Lab Activity 2 – February 11, 2019 Objective: Use trypsin to detach the cells and count the cells using hemocytometer. Materials Hemocytometer Trypan blue 3 concentrations of cell suspension given by the instructor 1 cell suspension made by your group Media Trypsin Microscope Procedure 1. Take your cell culture plate. Follow the trypsinization protocol. 2. Make cell suspension in 1 ml media. 3. Take 20 µl of cell suspension from each of the three cell suspensions given by the instructor and the cell suspension made by your group. Pipette the cell suspension in a centrifuge tube, and add 20 µl of trypan blue to the cell suspension. 4. Count the live and dead cells. 5. Repeat Steps 3 and 4, three times for each cell suspension. 6. Record all the readings and report average and standard deviation for each cell suspension. 9 
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