PuraMatrix™ Peptide Hydrogel.pdf

SPC-354250-G rev 3.0 BD PuraMatrix Peptide HydrogelCatalog No.354250 Guidelines for Use FOR RESEARCH USE ONLY.NOT FOR CLINICAL,DIAGNOSTIC.OR THERAPEUTIC PROCEDURES.NOT FOR USE IN HUMANS.BD Biosciences Discovery Labware Two Oak Park,Bedford,MA 01730,tel:877.232.8995,fax:800.325.9637, For Research Use Only.Not for use in clinical,diagnostic,or therapeutic procedures.Not for use in humans.1 SPC-354250-G rev 4.0TABLE OF CONTENTS Page INTENDED USE.2 MATERIALS PROVIDED.3 SUPPLEMENTAL/OPTIONAL MATERIALS(NOT SUPPLIED).3 PRECAUTIONS.3 GENERAL USE.4 PROCEDURES FOR USE.4 A)Surface Plating of Adherent Cells in Cell Culture Plates.4 B)Surface Plating of Adherent Cells in Cell Culture Insert.5 C)ECM Preparation for Supplementation of BD PuraMatrix Peptide Hydrogel.6 D)3D Cell Encapsulation in Cell Culture Plates(with or without ECM protein supplementation).8 E)3D Cell Encapsulation in Cell Culture Inserts(with or without ECM protein supplementation).8 F)Cell Drop Culture Protocol.11 G)Cell Recovery for Sub-Culturing or Biochemical Analyses.12 H)Fluorescent Staining of Samples Cultured with BD PuraMatrix Peptide Hydrogel.13 I)Tips for In Vivo Delivery of Cells and/or Bioactive Molecules.15 STABILITY.16 TECHNICAL SUPPORT.16 CUSTOMER SERVICE.16 For Research Use Only.Not for use in clinical,diagnostic,or therapeutic procedures.Not for use in humans.2 SPC-354250-G rev 4.0INTENDED USEBD PuraMatrix Peptide Hydrogel(BD PuraMatrix)is a synthetic matrix that is used to create defined three dimensional(3D)microenvironments for a variety of cell culture experiments.To achieve optimal cell growth and differentiation,it is necessary to determine the appropriate mixture of this material and bioactive molecules(e.g.,growth factors,extracellular matrix(ECM)proteins,and/or other molecules).BD PuraMatrix consists of standard amino acids(1%w/v)and 99%water.Under physiological conditions,the peptide component of BD PuraMatrix self-assembles into a 3D hydrogel that exhibits a nanometer scale fibrous structure with an average pore size of 50-200 nm.The hydrogel is readily formed in a culture dish,multiwell plate,or cell culture insert.BD PuraMatrix provides the benefits of 3D cell culture while allowing more straightforward 2D plating.Many cell types form appropriate 3D morphologies when plated on the surface of BD PuraMatrix,which simplifies the work required to develop 3D in vitro model systems compared with cell encapsulation or sandwich culture systems.Research has shown that cells forming 3D structures often resemble their in vivocounterparts more closely when compared to cells grown in 2D.BD PuraMatrix allows defined microenvironments to better recapitulate the in vivo milieu,which should improve the predictive quality of results obtained from assays conducted in a high-throughput context.The hydrogel has been shown to support the differentiation of hepatocyte progenitor cells1,rat pheochromocytoma cells(PC12)2 and hippocampal neurons3,as well as enabling endothelial cell tubulogenesis4.Studies have also demonstrated that BD PuraMatrix supports the attachment of a variety of primary(e.g.,neuronal,fibroblast,and keratinocyte)and transformed(e.g.,MG-63,SH-SY5Y,HEK293,and NIH3T3)cell types5,6.Other potential applications include stem cell proliferation and differentiation,tumor cell migration,and in vivo analyses of tissue regeneration.BD PuraMatrix is biocompatible,resorbable,and devoid of animal-derived material and pathogens.For in vivo studies in animals,the soluble material can be injected and will subsequently form a 3D hydrogel upon contact with the physiological environment.References1.Semino,C.E.,et al.,Differentiation 71:262(2003).2.Holmes,T.C.,et al.,PNAS USA 97:6728(2000).3.Semino,C.E.,et al.,Tissue Engineering 10:643(2004).4.Narmoneva,D.,et al.,Biomaterials 26:4837(2005).5.Zhang,S.,et al.,Biomaterials 16:1385(1995).6.Thonhoff,J.R.,et al.,Brain Research 1187:42(2008).For additional information,visit our website at: Research Use Only.Not for use in clinical,diagnostic,or therapeutic procedures.Not for use in humans.3 SPC-354250-G rev 4.0MATERIALS PROVIDED BD PuraMatrix Peptide Hydrogel(BD Cat.No.354250)packaged in one vial containing 1%solution(w/v)of purified synthetic peptide(5 ml per vial)SUPPLEMENTAL/OPTIONAL MATERIALS(NOT SUPPLIED)Tissue culture media for cells of interest 20%sucrose in distilled water(dH2O),sterile filtered(prepare 10%sucrose stock by diluting with sterile water)ECMs,growth factors,and cytokines BD Falcon Cell Culture Inserts and BD Falcon Cell Culture Plates For information about available products from BD Biosciences,visit our website at:Hints For Optimal Results with BD PuraMatrix Peptide Hydrogel PRECAUTIONS GELATION:Since the gelation of this material is initiated by salt concentrations?1 mM,this process may be promoted by salt-containing buffers and/or media.Therefore,do not combineBD PuraMatrix with salt-containing buffers and/or media until gelation is desired.Once the gel is formed,any subsequent mixing of the sample will disrupt the structure of the hydrogel.VISCOSITY:Decrease the viscosity of the stock solution(1%w/v)by vortexing or sonication for 30 minutes in a bath sonicator every time the stock is used.If air bubbles are present,centrifuge an aliquot of the stock in a 1.5 ml microtube at full speed.pH and OSMOLARITY:Since BD PuraMatrix exhibits a pH of 2-2.5,work quickly to minimize the amount of time that cells are in contact with the material prior to the addition of media when performing cell encapsulation experiments.Addition of isoosmotic sucrose to the cell suspension as well as to the BD PuraMatrix,will help protect the cells until the pH is normalized by equilibration with tissue culture media.For details,see Parts D and E(Cell Encapsulation Protocols).HANDLING:-Since BD PuraMatrix forms a relatively soft fibrous matrix,it is necessary to handle the material very carefully when performing media changes.Do not use vacuum aspiration to remove media from above the hydrogel.Avoid direct contact with the hydrogel.-When adding media and/or other components to the sample,place the end of the pipette tip towards the top of the culture vessel wall.-When using culture vessels with large surface areas(e.g.,?35 mm dishes),perform fluid handling steps with extreme care to avoid disruption of the hydrogel.For Research Use Only.Not for use in clinical,diagnostic,or therapeutic procedures.Not for use in humans.4 SPC-354250-G rev 4.0GENERAL USE See table on page 15 to determine appropriate volume for desired BD Falcon Cell Culture Insert or Multiwell Plate format.BD PuraMatrix can be used in the undiluted form at a concentration of 1%(w/v)or diluted as described in PROCEDURES FOR USE.Concentrations below 0.5%are suitable for many applications.For example,we recommend 0.25%for surface plating of most cell types,and a final concentration of 0.15%for encapsulating neurons and endothelial cells.To supplement the hydrogel with ECMs,growth factors,and/or other bioactive molecules,refer to Part C(ECM Supplementation Protocols).If using serum-free media for culturing cells,it may be necessary to deactivate the trypsin following cell isolation by trypsinization.IMPORTANT:To achieve optimal cell growth and differentiation,it is necessary to determine the appropriate mixture of BD PuraMatrix Peptide Hydrogel and bioactive molecules(e.g.,growth factors,ECM proteins,and/or other molecules).In general,it may be preferable to add growth factors to the media rather than encapsulate them in the hydrogel for most applications.PROCEDURES FOR USEA)Surface Plating of Adherent Cells in Cell Culture Plates Note:Many cell types that are plated on the surface of BD PuraMatrix can assume the appropriate and expected 3D structures(e.g.,hepatoycyte spheroids,embryonic stem cell colonies and embryoid bodies,neuronal cell will branch).The 2D surface plating method is an ideal tool for screening both morphological and functional characteristics of any given cell type.BD PuraMatrix Preparation and Gel Formation1.Decrease the viscosity of the BD PuraMatrix stock solution(1%w/v)by vortexing or by sonication in a bath sonicator as described above.Prepare the appropriate volume ofBD PuraMatrix in a microtube by diluting the stock with sterile dH2O.2.Prepare a sufficient volume of the desired concentration of BD PuraMatrix(e.g.,250 l for a 24-well plate or 50 l for a 96-well plate)by dilution with sterile dH2O.Add to the surface of the well and promote gelation by carefully and slowly adding medium to each well(e.g.,500 l for a 24-well plate or 100 l for a 96-well plate).3.Place plate in an incubator for 30-60 minutes to complete the gelation of the BD PuraMatrix.For higher percentage hydrogels,30 minutes is sufficient;for 0.15-0.25%hydrogels,a 1-hour equilibration is preferable.After the hydrogel has assembled,carefully change the medium as described above.Change the medium two times over a period of 1 hour to equilibrate the growth environment to physiological pH.If necessary,the equilibrated sample can be stored overnight at 37C after the final media change.For Research Use Only.Not for use in clinical,diagnostic,or therapeutic procedures.Not for use in humans.5 SPC-354250-G rev 4.0Cell PlatingTrypsinize cells and spin down desired number(typically 4-16x104 cells/cm2 final concentration for most cell types).Resuspend cells in tissue culture media at an appropriate concentration of cells in a final volume of 200-350 l.Carefully add the cell suspension to the top of the hydrogel.B)Surface Plating of Adherent Cells in Cell Culture Inserts The following protocol is based on the use of a BD Falcon 24-well Cell Culture Insert(BD Cat.No.353095)and a BD Falcon Cell Culture Insert Companion Plate(BD Cat.No.353504).BD PuraMatrix Preparation and Gel Formation1.Decrease the viscosity of the BD PuraMatrix stock solution(1%w/v)by vortexing or by sonication in a bath sonicator as described above.Prepare the appropriate volume ofBD PuraMatrix in a microtube by diluting the stock with sterile dH2O.We recommend a total volume of 100 l per cell culture insert at a final concentration of 0.25%for most cell types.Mix by gentle pipetting.2.Place desired number of cell culture inserts in the insert companion plate.Add 250 l of media to the lower chambers of each 24-well insert by pipetting down the side of the outer well between the insert and the well.Pipet carefully to avoid bubbles under the insert.The media should just touch the bottom of the insert.See Table on page 15 for cell culture insert volume recommendations.3.Pipet 100 l of diluted BD PuraMatrix into the center of each of the inserts.Wait at least 30 minutes for the BD PuraMatrix to gel.4.VERY CAREFULLY layer 400 l media onto the gel with a pipet tip(use a P200 pipetter and add 200 l(two times)for smaller droplet size)by running it slowly down into the inner wall of the insert.5.Follow steps 10-13 in Part E to complete pH equilibration of the BD PuraMatrix prior to cell plating.Cell Plating1.Trypsinize cells and spin down desired number(typically 4-16x104 cells/cm2 final concentration for most cell types).Resuspend cells in tissue culture media at an appropriate concentration of cells in a final volume of 200-350 l.2.Remove the media above the hydrogel and from the well as described above.3.GENTLY pipet the cells into the inserts using a P200 pipetter,being careful not to disturb the hydrogel.4.Add 700-900 l of media below each insert.5.For further cell feeding,change the media VERY GENTLY approximately every two days as described above.For Research Use Only.Not for use in clinical,diagnostic,or therapeutic procedures.Not for use in humans.6 SPC-354250-G rev 4.0C)ECM Preparation for Supplementation of BD PuraMatrix Peptide Hydrogel Note:Prior to use,decrease the viscosity of the BD PuraMatrix stock solution(1%w/v)by vortexing or by sonication in a bath sonicator for 30 minutes every time the stock is used.If air bubbles are present,centrifuge an aliquot of the stock solution in a 1.5 ml microtube.ECM Supplementation for Surface Plating of CellsFibronectin Protocol:(Since fibronectin is not soluble in the absence of salt,it must be coated onto the fibers).1.Resuspend 5 mg BD Fibronectin(BD Cat.No.356008)in 5 ml sterile dH2O.2.Dialyze overnight at Room Temperature(RT)using 10,000 MW cutoff Slide-A-Lyzer(Pierce Cat.No.66453),against dPBS without Ca2+and Mg2+.3.Prior to addition of fibronectin,mix BD PuraMatrix thoroughly with gentle pipetting and add to well of appropriate plate at desired concentration.See Table on page 15 for suggested volumes.4.Gel BD PuraMatrix by adding Fibronectin/PBS solution(1 mg/ml)on top of the BD PuraMatrix for 60 minutes.5.Carefully rinse with cell culture media for 5 minutes,aspirate and then carefully add cells to the top of the matrix.Collagen I Protocol:(Collagen I is soluble in 1 mM HCl and thus can be incorporated into theBD PuraMatrix before gelation):1.Dialyze collagen I(stock solution in acetic acid,BD Cat.No.354236)using 10,000 MW cutoff Slide-A-Lyzer against 1 mM HCl overnight at 4oC.2.Add the required amount of collagen I/HCl to BD PuraMatrix and mix with gentle pipetting.3.Add to well.4.Gel with cell culture media by adding media carefully to side wall of cell culture insert or well of plate.5.Once gel has formed,remove media carefully with a pipet and change two more times over the next one hour to equilibrate the BD PuraMatrix to physiological pH prior to plating the cells on top of the matrix.For Research Use Only.Not for use in clinical,diagnostic,or therapeutic procedures.Not for use in humans.7 SPC-354250-G rev 4.0Laminin Protocol:(Laminin is provided in 0.05 M Tris-HCl,0.15 M NaCl,pH 7.4).1.Low concentration laminin Laminin,mouse(BD Cat.No.354232)and Ultra-pure Laminin,mouse(BD Cat.No.354239).a.Mix with cell culture media at the desired concentration.b.Carefully add the mixture directly on top of BD PuraMatrix(prepared in the presence or absence of cells)in the cell culture insert or plate to promote gelation.2.Laminin/Entactin,High Concentration(HC),10 mg/ml(BD Cat.No.354259).Add required volume of HC Laminin directly to BD PuraMatrix.At dilutions of HC Laminin that result in a final concentration of 1 mM salt,gelation of BD PuraMatrix will not occur.To induce gelation after the addition of Laminin,add cell culture media and equilibrate the pH as described in the collagen I protocol.ECM Supplementation of BD PuraMatrix for Cell EncapsulationDetailed instructions for cell encapsulation in BD PuraMatrix can be found in Parts D and E.These should be followed,with the modifications detailed below,to introduce ECMs into the cell encapsulation mixture.For BD PuraMatrix concentrations 0.25%,a cell culture insert is recommended to facilitate media changes and ensure that the hydrogel is not disrupted.BD PuraMatrix concentrations below 0.25%are recommended for the encapsulation of neurons and endothelial cells.With other cell types,testing a range of hydrogel c