This README.txt file was generated on 2022-10-10 by María Inmaculada García Briega ------------------- GENERAL INFORMATION ------------------- Title of Dataset: Stability of Biomimetically Functionalised Alginate_data Author Information Principal Investigator: María Inmaculada García Briega; 1,2; inmagarciabriega@gmail.com; https://orcid.org/0000-0003-0811-2632 Associate or Co-investigator: Joaquín Ródenas-Rochina; 1; jrodenasr@gmail.com; https://orcid.org/0000-0001-5764-207X Luis Amaro Martins; 1; luisamaromartins@gmail.com; https://orcid.org/0000-0001-8648-6459 Senentxu Lanceros-Méndez; 3,4,5; senentxu.lanceros@bcmaterials.net; https://orcid.org/0000-0001-6791-7620 Gloria Gallego Ferrer; 1,2; ggallego@ter.upv.es; https://orcid.org/0000-0002-2428-0903 Amparo Sempere; 6,7; sempere_amp@gva.es; https://orcid.org/0000-0001-6727-3343 José Luís Gómez Ribelles; 1,2; jlgomez@ter.upv.es; https://orcid.org/0000-0001-9099-0885 Affiliations: 1- Centre for Biomaterials and Tissue Engineering (CBIT) Universitat Politècnica de València, 46022 Valencia, Spain 2- Biomedical Research Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 46022 Valencia, Spain 3- Centre of Physics, Universidade Do Minho, 4710-057 Braga, Portugal 4- BCMaterials, Basque Centre for Materials, Applications and Nanostructures, UPV/EHU Science Park, 48940 Leioa, Spain 5- IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain 6- Grupo de Investigación en Hematología, Instituto de Investigación Sanitaria La Fe (IISLAFE), 46026 Valencia, Spain 7- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto Carlos III, 28029 Madrid, Spain Date of data collection: From 2021-06-01 to 2022-09-30 Geographic location of data collection: Centro de Biomateriales e Ingeniería Tisular, Universitat Politècnica de València, Valencia, Spain (39.478006, -0.333463) Information about funding sources or sponsorship that supported the collection of the data: Spanish State Research Agency (AEI) through PID2019-106099RB-C41/AEI/10.13039/501100011033 and PID2019-106099RB-C43/AEI/10.13039/501100011033 project. Spanish Ministry of Science, Innovation and Universities through Grant N° PRE2020-092326 awarded to Mª Inmaculada García-Briega General description: The excel documents contain the crude numerical data of all the quantitative outputs of the article "Stability of Biomimetically Functionalised Alginate Microspheres as 3D Support in Cell Cultures" published October-November 2022 in the journal Polymers (MDPI). The title of each file identifies which test the contained data corresponds to. The data is obtained in order to characterise the biomaterials developed for the BIOMICROGEL project prior to their use in cell culture. Keywords: alginate; microspheres; stability; layer by layer; multiple myeloma -------------------------- SHARING/ACCESS INFORMATION -------------------------- Open Access to data: Open Date end Embargo: non required Licenses/restrictions placed on the data, or limitations of reuse: Creative Commons (CC-BY) Citation for and links to publications that cite or use the data: not available yet Links/relationships to previous or related data sets: no other related data available Links to other publicly accessible locations of the data: do not exist -------------------- DATA & FILE OVERVIEW -------------------- File list: - Blyscan_paper: It contains the results of heparin quantification of our microspheres. These results are in absorbance and are processed in the excel until they are converted into concentrations of the glycosaminoglycan in question. - ninhidrin_assay: It contains the results of the quantification of the poly-L-lysine in our microspheres. These results are in absorbance and are processed in the excel into concentrations of the biopolymer in question. - resultados_analizados: It contains the results of the Picogreen assay, which quantifies the total DNA in each of the cell culture conditions. These results are in absorbance (signal given by all the DNA in the sample) and are processed in excel to convert them into the number of cells in each sample in our work. - Day0:It contains the results of the equilibrium swelling test on the day of the start of the study. This test compares the diameters of the 3 types of study microspheres in different liquid media. These results are in micrometres, and are the Feret diameters of all microspheres recorded in each condition. - Day1: It contains the results of the wet equilibrium test on the day 1 of the study. This test compares the diameters of the 3 types of study microspheres in different liquid media. These results are in micrometres, and are the Feret diameters of all microspheres recorded in each condition. - Day14: It contains the results of the wet equilibrium test on the day 14, the end of the study. This test compares the diameters of the 3 types of study microspheres in different liquid media. These results are in micrometres, and are the Feret diameters of all microspheres recorded in each condition. - evolución peso: It contains the weight loss results of the uncoated microspheres from the work with water evaporation. This data is in milligrams and is processed to know the water content in the microspheres of the article. - diámetros: It contains the Feret diameters of the different conditions tested in the microfluidic system. These conditions are pairs of flow rates of the batch and continuous media in micrometres and are the Feret diameters of all measured microspheres for each pair of microfluidic flow rates. - ftir PAPER:It contains the results of the FTIR test conducted on all the microspheres obtained and the materials used for their manufacture. These values are wavelengths and their corresponding transmittance, with arbitrary units of measurement (a.u). - TGA_moLbL_PAPER: Includes the results of the thermogravimetric analysis performed on all the microspheres obtained and the materials used to manufacture them. These values include time, temperature and weight, as well as the data processing conducted in each condition in order to obtain the desired information together with the graphs used in the article. Relationship between files: no needed Type of version of the dataset: raw data in Day0, Day1, Day14, diámetros and ftir PAPER. In the rest of the documents, the data processed. Total size: 33,4 MB -------------------------- METHODOLOGICAL INFORMATION -------------------------- Description of methods used for collection/generation of data: - Microsphere Characterisation Optical microscopy, ImageJ imaging software and Excel and Graphpad statistical software were used to evaluate the diameter of the microspheres. Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) were used to determine their composition. The TGA parameters for the measurements were a heating rate of 10 °C/min from 25 to 1000 °C and a nitrogen flow rate of 50 mL/min. The samples analysis was carried out at temperatures above 200 °C, when no water remained in the microsphere. Blysscan (B1000 Blyscan kit from Bicolor, Beverly Hills, CA, USA) and ninhydrin colorimetric assays were used to quantify the amount of HEP and PLL in the coating, the first using the company protocol (https://www.biocolor.co.uk/ecm-assays/blyscan-glycosaminoglycan-assay) latter based on a protocol described by Algieri et al. (https://doi.org/10.1002/bab.1462.) Both colorimetric assays were performed in the Perkin Elmer Victor X3 Multilabel Placa Lector. - Equilibrium Swelling Equilibrium swelling is a quantitative method of determining the liquid media in which the synthesised microspheres maintain their cross-linked structure and remain insoluble. Their stability was tested in different culture media (RPMI 1640, DMEM R, DMEM W, PBS +/+, and PBS −/−) and in water at different pH to assess the influence of pH on cross-linking stability. This test also verifies that the coating allows the interior to liquefy by immersing all types of microspheres in EDTA, which is a calcium chelator. The test assesses variations in diameter in each condition by means of the Feret diameter, measured by ImageJ on optical microscopy images and GraphPad statistics. - In Vitro Assay An in vitro assay was performed on days 3 and 5 to test cell proliferation with and without microspheres. The culture platform comprised 60,000 cells of the RPMI 8226 cell line in 200 μL of RPMI 1640 culture under gentle stirring by a rotational shaker. There were three conditions: cells in suspension without microspheres, cells with the moPLL/ALG microgel, and cells with moCHI/HEP microgel. The percentage of micro-spheres in relation to the total volume was 7%, and the suspension was agitated by a commercial rotational shaker (±60° oscillation at 25 rpm). A PicoGreen assay was performed with a Quant-iT™ PicoGreen® dsDNA kit from Invitrogen to evaluate the culture’s cell proliferation (https://www.fishersci.es/shop/products/quant-it-picogreen-dsdna-assay-kits-dsdna-reagents/10398702) using the Perkin Elmer Victor X3 Multilabel Placa Lector Methods for processing the data: - In all the assays except in TGA and FTIR, the results are provided as mean ± standard deviation (SD). The row data was analysed, and outliers were identified using the ROUT method with a Q of 5%. After removing the outliers, the normality of the different samples was checked using the Shapiro–Wilk normality test with an alpha of 0.05. Unpaired T-Student tests (p-value = 0.05) were used to compare two single groups of data. An ordinary one-way ANOVA test (p-value = 0.05) was used for three or more groups to perform multiple comparisons between the column means when the normality test was passed. If this test was not passed, then the non-parametric Kruskal–Wallis test was used to compare this non-normal sample with other normal samples (p-value = 0.05) to perform multiple comparisons between the column means. GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA) was used for the statistical analysis. Differences among groups were stated in the article's figures as p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****) in normal and p ≤ 0.05 (#) in non-normal samples. - In FTIR, the figures were obtained adding some static value to all the transmitance data for a sample. This let us to graphicaly represent the FTIR spectra of all the samples and pristine materials in a single figure and recognise the different waveleght peaks to relate them with the composition of the samples. The increments made are recorded in the excel file of ftir PAPER. - In the TGA assay, more complex data processing was made. The equipment that performs the heating sweep has an associated software (STARe Software Version 13.0), that let us to obtain a document with the drop in weight of the sample as a function of temperature and the derivative of this temperature drop with respect to time, which is analysed in Excel. For comparison, the derivative is normalised by the dry weight of the sample (T at 200 ºC). We take advantage of the fact that alginate has a characteristic degradation, which can be modelled with the sum of two normal curves between 200 and 400ºC, to extrapolate with the use of normals and the areas of the curves the percentage of weight corresponding to the coatings. All these data are in the excel document for all the replicates and all the microspheres analysed. Software- or Instrument-specific information needed to interpret the data, including software and hardware version numbers: - Graphpad 8 or more to analyse all the statistics (cleaning outliers, normality tests and statistical comparisions between samples). - ImageJ or Fiji software for the analysis of all the images of the microspheres to obtain their diameter in the required tests (last version available online for free). - Excel software (version 16051.15629.20156.0 from Microsoft) for some data manipulation - STARe Software Version 13.0 for TGA data obtention. Standards and calibration information, if appropriate: Environmental/experimental conditions: The temperature was always between 20-25ºC Describe any quality-assurance procedures performed on the data: -------------------------- DATA-SPECIFIC INFORMATION -------------------------- - Blyscan_paper Number of sheets and explanation: 1st sheet = plate absorbances without more information 2nd sheet = protocol of the Perkin Elmer Victor X3 Multilabel Placa Lector to perform this assay 3rd sheet = data processing step by step Number of variables: 1 (absorbance of the sample) Number of cases/rows: we have 3 biological specimens for each of our 2 conditions in the experiment (control and (CHI-HEP)3-AGUA). In addition, each biological replicate has 3 technical replicates. Variable list, defining any abbreviations, units of measure, codes or symbols used: RB = Biological specimen of a condition control = uncoated alginate microspheres (CHI-HEP)3-AGUA = 3 bilayers coated alginate microsphere with Heparine and chitosan Missing data codes: Specialized formats or other abbreviations used: ug = micrograms uL = microliters mg = milligrams - ninhidrin_assay Number of sheets and explanation: 1st sheet = data processing step by step 2nd sheet = Calibation curve performed. Number of variables: 1 (absorbance of the sample) Number of cases/rows: we have 3 biological specimens for each of our 2 conditions in the experiment (control and P-A-P-A). In addition, each biological replicate has 3 technical replicates. Variable list, defining any abbreviations, units of measure, codes or symbols used: RB = Biological specimen of a condition abs = absorbance control = uncoated alginate microspheres P-A-P-A = 2 bilayers coated alginate microsphere with PLL and alginate Missing data codes: there is not missing data Specialized formats or other abbreviations used: ug = micrograms uL = microliters mg = milligrams uM = micromolar g = grams mo = microspheres PLL = Polylysine alg = alginate - resultados_analizados Number of sheets and explanation: 1st sheet = row data and step by step processing of the data of the empty tubes where the cell culture was done 2nd sheet = row data and step by step processing of the data of the content of the tubes where the cell culture was done Number of variables: 1 (absorbance of the sample) Number of cases/rows: we have 4 biological specimens for each of our 3 conditions in the experiment (control without microspheres, cells with Polylysine-alginate coating and cells with heparine-chitosan coating) and our 2 data acquisition time (3 and 5 days). In addition, each biological replicate has 3 technical replicates. Variable list, defining any abbreviations, units of measure, codes or symbols used: LbL = Layer by layer = cell culture with Heparin-chitosan coated microspheres control = cell culture with polylysine-alginate coated microspheres Suspension = cell culture without microspheres Missing data codes: Specialized formats or other abbreviations used: std = standard Desvest = standard deviation ng = nanograms 3d = 3 days 5d = 5 days - Day0, Day1 and Day14 (3 different files, but all 3 have the same structure and kind of data) Number of sheets and explanation: 1st sheet to 9th sheet = row data of the alginate microspheres studied in the paper. 10th sheet = blank sheet to separate between types of microspheres sheets. 11th sheet to 20th sheet = row data of the chitosan-heparine coated microspheres studied in the paper. 21st sheet = blank sheet to separate between types of microspheres sheets. 22nd sheet to 31st sheet = row data of the polylysine-alginate coated microspheres studied in the paper. Number of variables: 7 (Area, Feret, feretX, feretY, FeretAngle, Minferet, media feret) Number of cases/rows: Each excel corresponds to a moment in the experiment. Within each file, there are the sheets which correspond to the 3 types of microspheres in the article in each of the conditions tested in this trial. In each condition and sample type, there was a variable number (it was somewhat difficult to control) of microspheres to measure and count (each row is therefore one microsphere detected and analysed). Variable list, defining any abbreviations, units of measure, codes or symbols used: Area = area of the microsphere analised Feret = maximum diameter obtained of the microsphere feretX = coordinate X of the center of the microsphere feretY = coordinate Y of the center of the microsphere FeretAngle = angle in which is the largest diameter Minferet = minimum diameter obtained of the microsphere media feret = mean of the maximum and minimum feret diameter moALG = alginate microspheres LbLChi = Feret_LbL CHI = heparine-chitosan coated microspheres LbLPLL = Feret_LBLPLL = polylysine-alginate coated microspheres Missing data codes: Specialized formats or other abbreviations used: EDTA = ethylenediaminetetraacetic acid RPMI = cell culture media for multiple myeloma DMEM R = Dulbecco’s Modified Eagle’s Medium High Glucose with Phenol Red DMEM B = Dulbecco’s Modified Eagle’s Medium High Glucose without Phenol Red DPBS +/+ = PBS +/+ = Dulbecco’s Phosphate-Buffered Saline with calcium and magnesium DPBS -/- = PBS -/- = Dulbecco’s Phosphate-Buffered Saline without calcium nor magnesium - evolución peso Number of sheets and explanation: 1st sheet = row data and step by step processing of the data Number of variables: 2 (time and weight) Number of cases/rows: we have 2 batches of material, and from each batch 3 biological samples were taken. For each time point studied, they were weighed once. Variable list, defining any abbreviations, units of measure, codes or symbols used: Missing data codes: Specialized formats or other abbreviations used: h = hours mg = milligrams gr = grams uL = microliters EWC = Equilibrium water content - diámetros Number of sheets and explanation: 1st sheet to 14th sheet = row data and some processing of the microspheres' diameter of each pair of fluxes (called condition) 15th sheet = summary sheet with the mean, standard deviation and error of all the microspheres for all the fluxes tested in the assay Number of variables: 6 (Feret, feretX, feretY, FeretAngle, Minferet, media feret) Number of cases/rows: between 50-60 rows/condition = between 50-60 microspheres measured/condition Variable list, defining any abbreviations, units of measure, codes or symbols used: Feret = maximum diameter obtained of the microsphere feretX = coordinate X of the center of the microsphere feretY = coordinate Y of the center of the microsphere FeretAngle = angle in which is the largest diameter Minferet = minimum diameter obtained of the microsphere media feret = mean of the maximum and minimum feret diameter Missing data codes: Specialized formats or other abbreviations used: mo = microsphere CF = Continuous flow/flux DF = Discontinuous fow/flux std = standard - ftir PAPER Number of sheets and explanation: 1st sheet to 3rd sheet = row data of the 3 types of microspheres studied in the paper. 4th sheet = row and processed data of the 3 types of microspheres studied in the paper and the graphs used in the paper 5th sheet = row and procesed data of the pristine materials used to obtain the 3 types of microspheres studied in the paper and the graphs used in the paper Number of variables: 2 (wavelenght and transmittance) Number of cases/rows: 1746/sample. We obtained the FTIR spectra for all the materials and microspheres of the atricle (sodium alginate, polylysine hydrobromide, chitosan, heparine, uncoated alginate microspheres, heparine-chitosan coated microspheres and polylysine-alginate coated microspheres) Variable list, defining any abbreviations, units of measure, codes or symbols used: moALG = alginate microspheres uncoated LbL-CHIHEP-3B-EN agua = C/H = heparine-chitosan coated microspheres LbL-PLLALG-2B = P/A = polylysine-alginate coated microspheres PLL = polylysine hydrobromide Alg = sodium alginate Hep = heparine Chi = chitosan Missing data codes: Specialized formats or other abbreviations used: cm = centimetres - TGA_moLbL_PAPER Number of sheets and explanation: 1st sheet to 3rd sheet = row and processed data of the alginate microspheres studied in the paper and the graphs obtained. 4th sheet to 6th sheet = row and processed data of the chitosan-heparine coated microspheres studied in the paper and the graphs obtained. 7th sheet to 9th sheet = row and processed data of the polylysine-alginate coated microspheres studied in the paper and the graphs obtained. 10th sheet to 11th sheet = row and processed data of the chitosan powder used to obtain the functionalisation of the microspheres 12th sheet to 13th sheet = row and processed data of the heparine powder used to obtain the functionalisation of the microspheres 14th sheet = row and processed data of the sodium alginate powder used to obtain the microspheres and their functionalisation. 15th sheet to 16th sheet = row and processed data of the polylysine used to obtain the functionalisation of the microspheres. Number of variables: 6 (time, temperature, weight, weight derivate with respect to time, weight percentage with respect to dry weight, weight derivate with respect to temperature normalised with dry weight) Number of cases/rows: Each sheet refers to one of the paper samples (sodium alginate, polylysine hydrobromide, chitosan, heparine, uncoated alginate microspheres, heparine-chitosan coated microspheres and polylysine-alginate coated microspheres) and has 5910 rows. All the rows conform the thermogravimetric spectra of one of the paper samples Variable list, defining any abbreviations, units of measure, codes or symbols used: igb_alg = moALG = alginate microspheres lbl-hep = moCHI/HEP = heparine-chitosan coated microspheres lbl-pll = moPLL/ALG = polylysine-alginate coated microspheres chi-puro = pristine CHI = chitosan powder hep-pura = pristine HEP = heparine powder PLL-pura = pristine PLL = polylysine hydrobromide powder t = time in seconds Ts = temperature in ºC Value = weight in milligrams y value = weight derivate with respect to time w(T)/w(200ºC) = weight percentage with respect to dry weight (1/vel)(1/w(200))dw/dT = dw(T)/dT*(1/w(200ºC)) = weight derivate with respect to temperature normalised with dry weight Missing data codes: Specialized formats or other abbreviations used: