Volume 4, Issue 1, 2011 Easy data template, analysis and presentation tools for complex multiparametric animal cell culture studies Maddaly Ravi, Associate Professor, Sri Ramachandra University, maddalyravi@hotmail.com Sai Keerthana W, UG student, Sri Ramachandra University, keerthanaw@gmail.com Madhumitha Haridoss, PG student,sri Ramachandra University, h.madhumitha@gmail.com Shwethaa Raghunathan, UG student,sri Ramachandra University, vrisha.rox.da.wrld@gmail.com Sowmya Thanikachalam, PG student,sri Ramachandra University, saumyasubramanium@gmail.com Abstract Cell culture experiments are important for biological education and research. Standardization of optimal conditions to obtain 3 Dimensional aggregates of a cell line was performed which involved studying the effects of several parameters with various combinations of the variables. Data records, analysis and interpretations become voluminous and complex with increasing variables. To simplify and semi automate this process, an excel sheet and a single table format was designed to represent the results of various combinations of the variables studied. These formats can be effectively used for science education and research as a methodology by all working with cell cultures. Introduction Animal cell cultures have become indispensable for a variety of uses and are integral to biological education globally. Cell cultures and experiments involving them are important components of undergraduate, graduate and research programmes in all fields of biological sciences. The reason is that once the required skills are acquired, cells in culture are reliable models for scientific studies. The culture techniques have evolved with simultaneous developments in supportive requirements for cultures such as media and supplements. [1] Cultured cells are being widely utilized for many studies including cell physiology, molecular biology and cancer research to mention a few. [2] Cell cultures are crucial for drug discovery and validation studies. The revolutionary Hybridoma technology to produce monoclonal antibodies is essentially a conglomeration of different cell culture methodologies. From cultures as 2 Dimensional models, the methodology has developed into culturing cells in a 3 Dimensional environment. These models are gaining importance in areas such as cancer research, tissue engineering etc., through better understanding of gene expressions and protein profile changes. From cells in cultures being the base for many products, they have now themselves become the products for direct applications. Cells can be cultured in variety of culture vessels that include flasks, plates and petri dishes. The choice of the culture vessel depends on the number of cells required, type of study, number of variables being tested and also the number of study point ranges within each of the study variable. When large cell numbers are required, culture flaks are convenient. When many variables are used in the study, such as the effects of more than one drug or various concentrations of one or more drugs, it is easier to work in culture plates. The 6, 12, 24 and 96 well plates where studies involving multiple variables can be conducted conveniently. For documentation, representation and analysis, it is imperative to have study design representative templates such as in the form of Tables in the word file, etc. The level of difficulty in such a word file, as a Table becomes increasingly difficult with increasing number of variables used in a study. For example, it can be tedious to type all the variables and parameters in each of the well area for a 24 well plate in a Table form of a word document. Multi well plates are also used to maintain cell cultures in different conditions. Parameters such as media used, seeding density, volume of culture etc. are varied for different cell lines for every culture that is set up. Creation of the tables and data entry to record the information for plate templates separately is time consuming. The software programme Excel has been used for various applications in the field of biology from analysis of statistical data to maintenance of lab records. [3] MS Excel was used for automating data entry for varied parameters used in animal cell culture and creating 1
templates for 6well, 12well and 24well cell culture plates. To automate and facilitate multi parametric analysis of data, a tool to tabulate diverse components involved in cell culture on a single platform was adopted as previously described. [4] The data was entered into an excel file to create a plate template. Programming the cells on the plate template was made such that the data entered in previously is automatically displayed in the plate template. Likewise, once the results are obtained, the need to represent the net result of a complex study in a single format is very useful for several reasons including significant reduction in the numbers of entities such as Tables/Graphs, etc and also for easier comparisons of the multiple variables. A single table where different parameters can be analyzed and represented was developed towards this. These innovative tools have applications for easy data presentation, analysis and interpretation and also as important components of teaching, learning and research at the undergraduate and graduate level practical sessions involving cell cultures. Methodology Cell culture plate layout designs with experimental multiparametric details as an excel file The parameters studied The parameters considered for the study include well identification number, volume of medium added for plating, concentration of agarose in the medium, the medium in which agarose was dissolved, presence or absence of conditioned medium and the cell seeding density. Data entry An excel sheet was opened and different columns were named according to the different parameters involved in various wells of a cell culture plate as represented in Figures 1 and 2. Creation of a template A template for 6 well, 12 well and 24 well plate was created using merge and centre for the selected cells. The border for the template was given in the following series of steps: The entire template was selected The selected template was left clicked on Format Cells was selected The tab Border in the dialogue box was chosen The options Outline and Inside and click on OK were selected 2
Figure 1. Data entry into excel cells for the fields well identity, agarose volume, % agarose composition, medium used for dissolving agarose, presence or absence of conditioned medium and seeding densities. Programming the excel sheet cells The cells corresponding to the culture plate wells were programmed to input data from the excel sheet using CONCATENATE function to merge data from the previously entered information at the top of the excel sheet. The programming was done by the following series of steps and represented in Figure 3. The cells to be programmed were selected The formula tab was clicked and concatenate formula was selected. It is a text function used to join several text strings into one text string and parenthesis is used to enter the formula for programming the cell. The formula starts with a double quotes and ends with a double quote: =CONCATENATE("",D2," of ",E2," % Agarose in ",F2," with a SD of ", H5," M (CM",G2,")") Figure 2. The template has been created using Merge and Centre Function and choosing Wrap Text option and the borders are framed using Format Cells 3
Figure 3. Programming of the Cells using CONCATENATE function to merge many text strings from different cells into single output cell. Conditional Formatting of the cells Conditional Formatting offers and easy way to apply formats that stay dormant until the values in the highlighted cells achieve a specified state as given in Figure 4. Here a tool was used to highlight cells with a certain color depending on cell value. The series of steps are as follows: The cells to be highlighted depending upon the condition were selected The option Conditional Formatting and select New Rule was clicked on The second option Format only cells that contain was selected and specific text in the dropdown box under Edit the rule description was subsequently selected and the value was entered. Format was clicked and different options such as number, font, border, fill etc., were used Ok was clicked to allow conditional formatting. On conditional formatting, different parameters are represented in different ways such as: The different cells containing PBS and RPMI are shown in different shades Cells with conditioned medium are shown by double underlining 4
Figure 4. Conditional Formatting of the programmed cells was used to change parameters in the format of the template to distinguish between different conditions in the cell culture plate Representation of analysed data In studies that involve series of experiments to standardize the 3D cell culture conditions, various variables and combinations of the variables have to be observed, data recorded and analyzed. For this purpose, a single template to analyze and represent all the parameters to record the results was developed. The parameters include concentration of agarose, culture surface area, types of culture medium, culture medium volumes, presence or absence of conditioned medium and various seeding densities. Thus the result entered in a single box is a representative of a combination of all these parameters as shown in Table 1. CONCENTRATION OF AGAROSE CM 0.25% 0.5% 0.75% +/ 6 12 24 6 12 24 6 12 24 PBS + 1 ml + 750 + 500 + 250 V O L U M E + 1 ml RPMI + 750 + 500 + 250 +/ X Y X Y X Y X Y X Y X Y X Y X Y X Y SEEDING DENSITIES Table 1. The template provides for various parameters including concentration of agarose, medium of cell suspension, volume of medium added, presence or absence of conditioned medium (CM) and seeding densities per well in a 6 well, 12 well & 24 well plate. 5
Results An excel file was developed in a series of steps with simple formulae to make data entry and record keeping simpler for multiparametric cell culture studies. Also, a single table format was developed which can serve as a template for simultaneous data entry, result representation and analysis of multiple parameters involved in cell culture studies. Discussion Cell cultures offer with excellent in vitro experimental models and should be well standardized for useful study results. [5] Standardization of the optimal cell types and the culture types/conditions require multiple experimental variables. Data analysis and interpretation is the most important and critical part of education and research. The raw data obtained from experiments should be presented in an organized manner so that useful information can be extracted from it. The excel template we describe here serves the required purpose of easy data entry along with maximum information as obtained in the final template. Also, the results obtained from all the variables/combinations of the variables could be entered into the single table format that we describe here. The advantages of these two tools (the excel template and the single table format) is requirement of less time, less confusion, less error and inclusion of maximum data. In addition, the formats are flexible to suit a variety of experimental circumstances. These inputs are designed to refine and distill the data so that students and researchers can glean interesting/pertinent information without the need to sort through voluminous data or a number of single entities such as graphs and tables. Conclusion Science education is a combination of imparting information, instilling the need for an analysis, asking the right questions and to choose the right approach thus contributing towards knowledge enhancement. In all areas of life sciences, basic research is a vital part of science education. Cell cultures owing to their immense potentials for both teaching and learning have become important components in science curricula globally. Data analysis and interpretation is the critical part of any research which involves multiple parameters and numerous variables. The cell culture plate template designed using MS Excel and table generated using MS Word that are described here present dense data in a simplified form. The generated data could be inspected, transformed and modeled with the useful and most pertinent information highlighted. These are intended to considerably reduce the complexity of data record keeping, presentation of the study design, analysis of results and sharing of the research findings. These tools can be very useful for both learning and teaching for practical education in biological sciences. References: [1] R. I. Freshney, A. R. Liss (1994) Culture of Animal Cells: A Manual of Basic Technique, 3rd ed., New York, Blackwell Publishing. [2] Adi F. Gazdar, Girard Luc, Lockwood William W., Wan L. Lam and John D. Minna (2010) Lung cancer cell lines as tools for biomedical discovery and research, J. Nat. Cancer Inst. 102, 1310 1321. [3] D. Z. Meyer, L. M. Avery (2009) Excel as a qualitative data analysis tool, Field Method. 21, 91 112. [4] M W. Lehman, C E. Herring (2003) Creating interactive spreadsheets to provide immediate feedback, J. Account. Educ. 21, 327 337. [5] J. H. Dunham, P. Guthmiller (2008). Doing good science: authenticating cell line identity, Cell Notice. 22, 15 17. 6