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1 & Basic&Laboratory& Materials&Science&and&Engineering& Biocompatible&Tests&of& Materials& M111& Stand: Aim: This lab serves as an introduction to testing the biocompatibility of materials by cell culture and fluorescence microscopy. Adherent cells will be cultured in vitro and fluorescently stained. Cell number should be determined and the adhesion of the cells on different samples should be examined by microscopy. 1. Basics What is cell culture? The cell culture lab Materials and Methods Cells in the light microscope Subcultivation /Passaging of adherent cells Cleaning the hood Cell detachment from the surface with trypsin: Counting cells Seeding cells on 2 different surfaces Staining fibroblasts Bibliography... 9

2 Safety Instructions for Biosafety Level 2 Laboratories You will be working in a Biosafety Level 2 laboratory, short S2 lab. In addition to the safety risks of hazardous (toxic, corrosive, mutagenic) chemical agents, in S2 labs pathogenic and genetically modified organisms are a source of danger. These organisms are infectious to humans when they are ingested or they penetrate through the skin with sharp items. For your own safety as well as others, you have to follow the safety rules strictly. I. Do not eat, drink, or chew gum in the lab. II. Always wear a lab coat and gloves. Change gloves immediately when contaminated. III. Do not touch your face, eyes, hair, mouth, etc. when you are in the lab, especially if you are wearing gloves. IV. First wash your hands and then sterilize (30 seconds long) before starting to work. After finishing your work, do it the other way around: first sterilize (30s) and then wash your hands, particularly before leaving the room. You might have pathogens on your hands, which must not be allowed to leave the room alive. V. Do not work alone and always follow the instructions of the course teacher. VI. Do not touch any equipment, chemicals, or other materials, especially culture vessels, until you are instructed to do so. VII. Keep your working place clean and tidy. Decontaminate it before and after the experiments. Spills have to be mopped up and disinfected immediately. VIII. Take care of infectious waste. Dispose of it properly in the appropriately marked containers. 2

3 1. Basics 1.1. What is cell culture? Cell culture is one of the major tools in molecular biology and overlapping research fields. Cell culture is a manifold method to study biochemical, metabolic, and physiological questions. It is the pre-stage used to test the effect of new materials and new drugs before animal testing, and it can be used to produce biological substances on an industrial scale (e.g. vaccines and proteins). The in vitro cultivation of cells means taking out single cell types from an organism, keeping them alive, and proliferating them in an artificial environment. There are different cell culture types: some cells can be grown in suspension, floating in the culture medium, but most cells are adherent and grow on a specialized surface as a monolayer. Cells derived directly from the organism are called primary cells. After subcultivation of a primary cell culture, the cells are called a cell line, because it is possible to keep them alive for more than one division cycle. Cell lines derived from primary cultures are finite. This means after several divisions they become senescent and lose the ability to proliferate (Hayflick limit). Immortal or continuous cell lines are usually derived from cancer cells or are transformed. For materials science, cell culture has evolved as an important field, especially when new materials are developed for medical applications where reliable risk assessments without animal experiments are needed. New materials are tested both for biocompatibility and also for their impact on cell behavior. Different properties of materials can either attract cells for adhesion, or repel them; it can even bring stem cells to develop into specialized tissue cells (so-called differentiation) The cell culture lab The culture conditions for eukaryotic cells are, as near as possible, adapted to the natural environment. To achieve physiological conditions, a specially equipped laboratory and materials are needed: a) Cell culture hood: the hood allows for a sterile working area. This is necessary for cell culture and experiments with living cells so as to avoid infections of the cell culture with bacteria and fungi. The hood maintains a constant, unidirectional flow of HEPA-filtered air (in our case a vertical flow) that protects the samples from dust particles, bacteria, viruses and other contaminants. b) Incubator: the incubator provides suitable environmental conditions, such as a stable temperature at 37 C and high humidity, to avoid evaporation of the medium. One important function is also the continuous maintenance of high CO 2 levels (5-10%) in the incubator. CO 2 is the corresponding reactant to the NaHCO 3 buffer component in the growth medium to prevent alkalinization due to secreted metabolic products. c) Water bath: cell culture products have to be kept cool when not in use, so before adding them to cells, liquids have to be pre-warmed. d) Inverted light microscope (phase contrast microscope) and a magnification of >100x e) Centrifuge to spin down cells f) Refrigerator (+4 C) and freezer (-20 C) for storage of media, reagents, drugs, antibiotics etc, deep freezer (-80 C) and a liquid storage nitrogen system for cryogenic storage of cell stocks. 3

4 g) Autoclave: sterilization of liquids, tools, and contaminated waste under high pressure at 121 C for >20min. h) Growth Medium: for different cell types, different growth media are needed. They all contain essential nutrients and substances to support proliferation and growth, such as amino acids, carbohydrates, vitamins and minerals. Cell medium contains a buffer system (mostly NaHCO 3 ) to maintain the physiological ph. The addition of Fetal Calf Serum (FCS) is essential as a source of growth factors and proteins, although the exact impact on the cells is not yet known. Also glutamine, an unstable but essential amino acid, has to be added as an alternative energy source for rapidly dividing cells. Additionally, antibiotics for fighting microbial contamination can be added, but ideally they are not needed when researchers use proper sterile working techniques. i) Sterile consumables like pipettes, centrifugation tubes, culture vessels, etc. 4

5 2. Materials and Methods 2.1. Cells in the light microscope Before cells are used for experiments, it is important to check their identity, viability, and their state of growth in order to have reproducible parameters. Mammalian cells are typically sized between 5 and 20 µm and can only be seen with a microscope. Unstained cells are usually viewed in the phase contrast mode, because transparent objects can be seen in more detail than in bright field mode. Place a culture flask containing cells under the microscope. Figure 1. Organelles of fibroblasts visible in phase contrast microscopy (scale bar 10µm) 2.2. Subcultivation /Passaging of adherent cells The provided cells are rat embryonic fibroblasts wild type (REF-52 wt). Fibroblasts are not completely differentiated cells of the connective tissue, and they synthesize collagen and major components of the extracellular matrix. They grow adherently in cell culture bottles with specially treated surfaces for ideal attachment. The growth of the cells in culture follows a standard growth curve (Figure 2). After a short period of slow growth (adaptation to the culture conditions), the proliferation is exponential, as long as there are enough nutrients and space available for expansion. If the growth area is crowded with spread cells, we talk about a confluent layer. Then proliferation is stopped by so-called contact inhibition. To keep cells in optimal growing conditions, they are subdivided before 100% confluency is reached (best between 70-80% confluency). 5

6 Figure 2. Characteric growth curve for cells in in vitro cultivation. After the initial lag phase, logarithmic proliferation starts until contact inhibition reduces proliferation activity. The best time for subcultivation is before the stationary phase is induced. Protocol for subcultivation: All solutions and materials that come into contact with cells must be sterile. Use proper sterile technique (have a look at the attached question table) and work in the hood. Materials: 70% ethanol (EtOH), pipettor, pipettes (10ml, 5ml, and 1ml), liquid waste container, pre-warmed complete growth media, pre-warmed PBS, trypsin solution, 15ml centrifugation tube, fresh culture flask Cleaning the hood Switch on the hood and put the glass front into the right position. Disinfect the materials and the working area exhaustively with 70% EtOH. Every item which is taken from outside into the hood has to be disinfected as well. Arrange the materials in an uncluttered way (Figure 3) and loosen all lids when the EtOH has dried. For time reasons, please skip to Cell detachment from the surface with trypsin: Remove and discard the media from the culture flask. Wash the cells with 5 ml PBS, ph 7.4. Add 1 ml of trypsin to the cells and cover the cell layer completely; place the flask in 37 C incubator for 2 minutes. Check the detachment with a microscope; you may need to rock the vessel gently to get complete detachment of the cell layer. Add 2ml of pre-warmed complete medium to the cells and disperse the medium by pipetting. Transfer the cell suspension to a 15 ml centrifugation tube and centrifuge at 800 x g for 5 min. Resuspend the cell pellet in pre-warmed complete growth medium. Pipet up and down to achieve a homogenous suspension of single cells. 6

7 Figure 3. Example of the organization of a working area in a hood. In the center is the clear working area. Materials are ordered around this area. Tools needed often should be easily reachable by hand. Gibco Cell Culture Basics handbook, Life Technologies Counting cells Besides viability and state of growth, an important factor for cell experiments is the cell concentration. Depending on the experimental setup, a defined number of cells per ml or cells per cm 2 material surface is needed to ensure the reproducibility of the experiments. Crowded cells reduce their spreading on the surface, which can be a judging factor. If cells are too diluted, they will be missing external input, like growth factors secreted from neighboring cells. Adhesion and proliferation can then be delayed, and lead to a misinterpretation of results. Materials: Neubauer counting chamber, cell suspension, pipette 20 µl, pipette tips, hand counter, calculator. The Neubauer counting chamber (Hemocytometer) is the most common method used for counting cells. It is a thick crystal slide with a counting grid engraved in the central area (Figure 4, left). The grid has 3x3 squares (side length 1mm each); the squares in the corners are used for the cell counts. A cover slide is placed on top of the grid. The grid depth is 0.1mm. 7

8 Figure 4. Counting grid of the Neubauer chamber (left) and counting rule (right). Cells on the upper and left borders should be counted, while those on the lower and right borders should not be counted. Basic Hemocytometer Usage, Celeromics.com Clean the counting chamber and the cover slip thoroughly; it must be particle free. Breathe on the coverslip until it is foggy, and slip it on the chamber surface. If the coverslip is placed correctly on the chamber, Newton s Rings can be observed. Pipette the cell suspension up and down before taking up a thoroughly mixed cell suspension. Place the filled pipette directly at the edge of coverslip. Slowly fill the gap between the coverslip and the counting chamber and let capillary forces distribute the suspension evenly over the grid. Count the cells in the 4 large squares with the help of the hand counter and stick to the counting rules (fig. 3 right). Calculate the cell concentration. The ideal cell concentration lies between 250,000 cells/ml and 2.5 million cells/ml to have a reliable counting result. If it is too low or too high the sample has to be either concentrated or diluted, and then re-counted Seeding cells on 2 different surfaces For time reasons, you will receive a prepared cell solution. Two different materials should be tested. Place the materials in 6-well plates and sterilize them for 2 min with 70% EtOH. Wash the samples twice with 4 ml PBS. Seed 30,000 cells/well in 4ml growth medium on the samples. Take care that the materials are completely covered with medium and that the cells are evenly distributed. Close the plate with the lid and place it in the incubator Staining fibroblasts One of the standard techniques for seeing cells better under a microscope is staining with dyes. Different dyes can stain different components of the cell, such as nucleus, cell wall, cytoplasm, etc, or they can be marked with antibodies coupled to fluorescent markers. It is also possible to get genetically modified cells that produce fluorescent proteins. Even with non-transparent adhesion surfaces, 8

9 such as metals or ceramics, cells can be observed, when fluorescently marked, at least when an upright fluorescence microscope is used. CAUTION: Wear blue nitrile gloves! The staining solutions contain DMSO. Staining the nucleus and the cytoplasm: The fluorescent dye Hoechst is used to stain the nucleus of cells. Hoechst intercalates into the double helix of the DNA and emits blue fluorescence (450nm) when excited with UV-light (350nm). Dilute the staining solution 1:1000 in the provided sample plates from 2.4. Let it incubate for at least 20 min at 37 C in the incubator. Keep the plates as long as possible in the dark to avoid early fading. Evaluate the material properties of the samples based on cell morphology and the number of adherent cells. Count the cells of 5 visual fields. 3. Bibliography Alberts et al.: Molecular Biology of the Cell, German Edition, Wiley-VCH Verlag 2003 Schmitz: Der Experimentator Zellkultur, Spektrum Akademischer Verlag 2011 Gibco Cell Culture Basics handbook, Life Technologies Basic Hemocytometer Usage, Celeromics.com 9

10 Attachment: Gibco Cell Culture Basics handbook, Life Technologies 10