Water permeability parameters of dermal fibroblast employed in tissue engineering in subzero tempera

530Science in China Ser. E Engineering and Materials Science 2005 Vol.48 No.5 530- -537Water permeability parameters of dermal fibro-blast employed in tissue engineering in subzerotemperaturesWANG Xin', CHENG Qikang', GAO Cai', YANG Pengfei', HUA Tsechao',DENG Chenliang', YANG Guanghui?, CUI Lei, LIU Wei?, CAO Yilin2,ZHAO Tingchang3 & SUN Fuzai?1. Institute of Cryomedicine and Food Refrigeration, Shanghai University of Science and Technology,Shanghai 200093, China;2. Shanghai Research & Development Center of Tssue Engineering, Shanghal 200235, China;3. Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beiing 100094, ChinaCorrespondence should be addressed to Hua Tsechao (email: tchua @ sht 63.net)Received May 13, 2005Abstract Fibroblast is a crucial kind of cell in the construction of the tissue engineereddermal equivalent. In order to optimize the cryopreservation protocols of the tissue-engineered dermis, the characteristics of dermal fibroblast in subzero temperatures arerequired, which include the water permeability of the cell membrane and the apparentactivation energy. Using the differential scanning calorimeter (DSC)， the volumetricshrinkage during freezing of human dermal fibroblast suspensions was obtained at thecooling rate of 5C . min~1 in the presence of extracellular ice. To ensure the presence ofextracellular ice, a small quantity of ice nucleation bacteria (INA bacteria), pseudomonassyringae was added in the samples. And based on the Karlsson's model, a nonlinear-least-squares curve ftting technique was implemented to calculate the cryogenicparameters. At the reference temperature TR(= 0C), the water permeability of membraneLp。= 0.578 μm. min-' . atm 1 and the apparent activation energy ELp= 308.8 kJ .mor-'.These parameters were then used to simulate water transport of filbroblast duringconstant cooling at rates between 0.01 一50 C .min-'. The simulation results wereanalyzed to predict the amount of water left in the cell after dehydration and the“optimalcooling rate" for fibroblast cryopreservation. For the dermal fibroblast with DMEM solution,a cooling rate of 4.6C . min^ 1 was optimal.Keywords: permeabilty characteristics, DSC, tsse engineered dermis, fibroblast.DOI: 10.1360/04ye0156In the construction of the tissue engineered中国煤花idermal fibroblastplays a crucial role!"! While the fibroblasts nee:DHC NM H Gng, a degadable:nthesize, and se-crete extracellular matrix in the three-dimensiCuusr oouti rustwwuCopynight by Science in China Press 2005.Water permeability parameters of dermal fibroblast employed in tissue engineering531scaffold commences disintegration over time. This may lead to an unusable product, ifproper preservation does not conduct. Cryopreservation could solve this problem. Thepossibility of the long-term banking of cells and tissues would provide a means for in-ventory control for both the manufacturer and end-user of the tissue product. Optimiza-tion of cryopreservation requires a quantitative understanding of the biophysical re-sponse in the target cell or tissue, e.g. the dermal fibroblast, during freezing. Clearly, theamount of water left within the cells during cryopreservation procedures is critical to thesuccess of the respective protocol, and therefore precise information about the rate andamount of water transport out of the cells at the subzero temperature is necessary.Cell membrane permeability to water could be reflected by the changes of cell vol-ume to extracellular conditions, and the water permeability parameters obtained at su-prazero temperatures are significantly different from those obtained at subzero tempera-tures. Therefore, to optimize a cryopreservation protocol, it is important to measure thepermeability of the cell membrane to water during freezing in the presence of extracel-lular ice and cryoprotective agents (CPA)4I. The differential scanning calorimeter (DSC)technique is one of the methods used to measure the water transport of the cell mem-brane during freezing, provided by Ramachandra et al. recently. The DSC technique isindependent of the size and shape of the cells being observed, and can be used to meas-ure the membrane permeability parameters at the subzero temperature and in the pres-ence of extracellular ice. The technique allows the measurement of the volumetric re-sponse of a large number of cells as opposed to single cell by standard cryomicroscopytechniques, and removes errors due to limitations of image analysis'3!. In this study, theDSC technique has been applied to measure the membrane permeability parameters ofdermal fibroblast, which is employed in the tissue engineered dermal replacement.1 Theoretical background of the DSC techniqueWater transport across a cell membrane during freezing in the presence of extracellu-lar ice has been modeled by Mazur4, and later modified by Levin et al. and Karlsson etal.l5,6]. The modified water transport model incorporates the presence of CPA on thevolumetric shrinkage response of cells during freezing as:dV_ LART(V-% -npa'qa)/wAH4VwP( 1_ 1-| lr(1)dT Bvw [ (%-V-nq%px)/w +(an +mp)RTR TEr[pa](1__ 1 11布= Lyp[Cpa]exp(2)where V is the cell volume at a subzero temperature T(K), Lpe is the permeability of themembrane to water at a reference temperature (TR中国煤化iarent activationenergy for the permeability process. R is the gasnt cooling rate. .YHCNMHGmolarAc is the effective membrane surface area for waur uauuspuil. vw i ue partialvolume of water. ns is the number of moles of solutes in the cell as calculated from ini-www.scichina.com532Science in China Ser. E Engineering and Materials Science 2005 Vol.48 No.5 530- -537tial cell osmolarity (C) and the total osmotically active cell water volume (ns =C:.(Vo-Vb)), where Vo and Vb are the isotonic (initial) and osmotically inactive cellvolume, respectively. 央is the disassociation constant for salt in water, and AHp is thelatent heat of fusion for water and is assumed to be constant in the temperature range ofinterest (0 to - 20°C) as 335 mJ.mg .nepa is the number of moles of salt or CPA. p is .the density of water. And Lp is an Arthenius function of Lpg and ELp.Based on the principle of DSC technique proposed by Ramachandra et al., the DSC-measured heat release can be translated to dynamic cell volume asVo-V(T) _ Aq(T)sc(3)V -VSqdsc .This equation can be rearranged to measure water transport data from the DSC meas-ured heat releases Oqdse and Aq(T)dsc asV(T)=V,--((7esVo -V%), .(4)Aqascwhere Oqasc is the total difference in heat release measured between the initial and finalcooling run, Qq(T)dsc is the fractional difference in heat release from phase change tem-perature (Tph) down to a subzero temperature (T).By fitting eqs. (1) and (2) to the data generated from eq. (4), the permeability of thecell membrane to water (Lpg and ELp) can be estimated.The DSC technique has been successfully applied to measure the membrane perme-ability parameters in a variety of biological systems, including mouse spermatozoa cellsuspensions', human lymphocyte cell suspensionso, as well as in liver tissue slices of afreeze tolerant wood frog'In this study, the DSC technique is used to measure the membrane permeability pa-rameters of human dermal fibroblasts, which are employed in the construction of tissueengineered dermal replacement.2 Materials and methods2.1 MaterialsHuman dermal fibroblast was provided by Shanghai Tissue Engineering R&D Center.Ice nucleation bacteria (INA) were provided by the Plant Protection Institute of ChineseAcademy of Agnicultural Science.2.2 DSCA DSC-Pyris Diamond (Perkin Elmer Corp中国煤化工) instrument wasused in this study.YHCNMHGCopyright by Science in China Press 2005.Water permeability parameters of dermal fibroblast employed in tissue engineering5332.3 Methods(i) Calibration of the DSC. To ensure the accuracy and repeatability of the experi-mental data, a set of calibration was' performed. The instrument was calibrated for tem-perature using hexane and water, and for energy using water only. Helium was used asthe purge gas (20 mL. min^'). Sigmoidal baseline for phase change was selected tominimize the baseline error during freezing.(i) Sample preparation.1) Human dermal fibroblasts were grown up in free suspension in the DMEM media(Dulbecco's Modified Eagle's Medium, Low Glucose), and spun down to higher celldensities to an average of (1.5+0.3)x10 cell. μL-.2) Viability assay. Cell viability was assessed by Trypan blue dye exclusion methodand found to be 95% for all experiments.3) For DSC experiments, the cell suspension along with 0.5 to 1 mg of ice nucleationbacteria (INA) was placed in standard aluminum sample pans and weighed. Care wastaken to ensure that the sample weight was always less than 15 mg.(il) DSC dynamic cooling protocols一slow-fast-slow (SFS) cooling protocol7l1) The sample initially at 4C was cooled at 5"C●min until the extracellular ice叫-cleated (in this experiment, about -5"C).2) At the time of nucleation, the sample was manually triggered to thaw at a warmingrate of 10°C . min~' such that Tph (- 2.2"C) was reached, and kept at this temperature for 5min (but not overshot). An equilibrating temperature of -2.2C was used so that theequibrium volume would be as close to the isotonic volume (V0} as possible, while stillmaintaining ice in the extracellular space.3) Then cool the samples to - -50°C at 5"C . min-', which caused the fibroblasts to un-dergo cellular dehydration. The lower curve in Fig. 1 corresponded to the heat releaseassociated with dehydration.4) The sample was re-equilibrated at Tph by thawing at 100"C . min and kept at Tphfor 2- 3 min, remaining careful to ensure that the sample remained nucleated with ice. .During this thawing process, a fraction of the cells rehydrates while other cels remainosmotically inactive or lysed due to freezing injury in step 3).5) In order to ensure that all of the cells become osmotically inactive due to freezinginjury, the sample was cooled at 200°C . min ! down2= 502中国煤化工6) Step 4) was repeated and all the cells wereTHCNMH Gnce all the cellshave compromised membranes, the intracellular water, proteins and salts were now con-www.scichina.com534Science in China Ser. E Engineering and Materials Science 2005 Vol.48 No.5 530- -537-2.366 7-10Step 7Step 3-20.74-20.41-i5-5-2.37Temperature/°CFig. 1. Superimposed heat flow thermograms obtained during step 3 and step 7 of the DSC cooling protocol forhuman dermal fibroblast cell suspension system, shown for a cooling rate of 5'C . rmin~sidered a part of the bulk solution.7) The sample was then cooled to - -50°C at 5C . min to measure the final heat re-lease. The upper curve in Fig.1 corresponded to this heat release. .3 Results3.1 Membrane permeability parametersThe total difference in the integrated heat release between the baseline (constant andsigmoidal) and the actual thermogram in the two cooling runs (step 3 and step 7) isshown in Fig. 1.Using the DSC Pyris Diamond software, the heat release measurements of interest,Oqse and Qq(T)dsc could be obtained. The initial volume of the fibroblasts in DMEMmedia was assumed to be the isotonic cell volume, Vo(5 105.5 μm° ) and the osmoticallyinactive cell volume, V%, was assumed to be 0.3631 Vo (1853.8 μm°) in DMEM mediaas reported earlier' 10. Then water transport data could be generated from DSC-measuredheat release readings using eq. (4). Fig. 2 shows the water transport data and simulationusing best-fit parameters in eqs. (1) and (2) at the cooling rate of 5"C .min~ .The unknown biophysical parameters of the model, parametric, can be determined bycurve-fitting the water transport model, eqs. (1) and (2), to experimentally obtain thewater transport data during freezing. An Isgnonlin function of Matlab 6.5 was imple-mented to calculate the membrane permeability parameters (Lpg and Erp) that best fit thevolumetric shrinkage data generated by DSC tec中国煤化工The best fit membrane permeability paramTYHCNM H Gal fibroblast inDMEM media are: Lpg = 0.578 μm . min 1. atm-' , Eup= 308.8 kJ.mol , R2 = 0.89.Copyright by Science in China Press 2005Water permeability parameters of dermal fibroblast employed in tissue engineering535Experimental data1.0Model simulation三0.60.4-0.2 t-5-10-15-20Temperature/*CFig. 2. The volumetric response of human dermal fibroblast as a function of subzero temperature obtained usingthe DSC technique, cooling rate: 5C . min 1.3.2 Simulations of water transport during freezingThe volumetric response of human dermal fibroblast at various cooling rates (at 0.01,1, 2, 5, 10, 20, and 50°C . min ') as a function of subzero temperature using the best fitmembrane permeability parameters could be obtained, following the methods stated byRamachandra et a.", and is shown in Fig. 3.In Fig. 3, for cooling rates ≤5°C . min~ , intracellular water could be almost trans-ported out to the extracellular space during freezing, and the end volume after watertransport was similar to the osmotically inactive cell volume, Vb , while for cooling rates0.950°C . min^日-20°C . min'10°C . min'0.75°C . min~2°C . min^ I1°C. min'一米一0.01°C . min^0.50.3Temperature/°C中国煤化工Fig.3. Volumetric response of human dermal fibroblast atYHC N M H Gnction of subzerotemperatures using the best fit membrane permeability parameters.www.scichina.com536Science in China Ser. E Engineering and Materials Science 2005 Vol.48 No.5 530- 537> 5°C . min-', intracellular water could be trapped within the cell, and the end volumewould be larger than V. With sufficient supercooling, this trapped water would ulti-mately form intracellular ice.Meanwhile, comparison between the experimental data and the model simulated dy-namic cooling response of human dermal fibroblasts at 5C . min~' was made, which isshown in Fig. 2. The differences in the water transport data between the experiment andthe simulation were not significant.3.3 The intracellular ice formation (IIF) and the optimal cooling rateThe optimal cooling rate for a certain kind of cell is affected by the volume of thewater trapped inside the cells at a given subzero temperature, e.g. - -30"C, where intra-cellular ice formation can occur by a homogeneous or volume-catalyzed nucleation. Thevolume of the water trapped inside the cells at -30C could be described as eq. (5).V 30一路W_ 30=-(5)V,-Vwhere V 30 is the end volume after water transport ceases (at -30°C), W_ 30 is the volumeof trapped water at -30°C.Table 1 shows the result of the calculated volume of trapped water at -30°C for avariety of cooling rates.Table 1 The volume of trapped water at -30C for a variety of cooling ratesCooling rate/C . min'V_30W_ 30/%0.010.38 Vo3.40.40 Vo6.30.41 Vo.350.43 Vo11.1100.62 Vo40.0200.79 Vo67.600.92 Vo86.7During freezing of cell suspension, the cooling rate that optimizes the freeze/thaw re-sponse of any cellular systems can be defined as the fastest cooling rate in a given mediawithout forming damaging intracellular ice (IIF)". Mazur defines I in embryos asdamaging and lethal if> 10% 一15% of the initial intracellular water is involved .And referring to the related researches of Ramachandra et al., we define the optimalcooling rate as the cooling rate at which≤10% of the initial osmotically active watervolume is trapped inside the cells at temperature,中国 煤化土ation shows thatthe optimal cooling rate in DMEM media for huMYHCNMH Gis 4.6C .min"',when a“two-step" cryopreservation protocol is applted.Copyright by Science in China Press 2005Water permeability parameters of dermal fibroblast employed in tissue engineering4 Conclusion(1) Using a new shape-independent differential scanning calorimeter (DSC) technique,the volumetric shrinkage during freezing of human dermal fibroblast suspension wasobtained at the cooling rate of 5 °C●min . By fitting the Karlsson' s water transportmode] to the experimentally obtained volumetric shrinkage data, the best fit membranepermeability parameters (Lpg and ELp) were determined.(2) These parameters were then used to simulate water transport in human dermal fi-broblast during constant cooling at rates between 0.01一50°C . min~，and predictionsabout the probability of IF and the optimal cooling rate were made.(3) Further research about the water permeability characteristics during freezing ofhuman dermal fibroblast needs to be conducted in the presence of cryoprotective agents(e.g., DMSO), and in a wider range of cooling rates.Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No.50376040), the Key Project of the National Natural Science Foundation of China (Grant No. 50436030), theNational 973 Project of China (Grant No. G1999054309), the National 863 Project of China (Grant No.2002AA205041), the Key Technologies R & D Project of Shanghai (Grant No. 00DJ14001-6), the EducationCommission of Shanghai and the Doctorial Start-up Project of Shanghai University of Science and Technology(Grant No. X 542).ReferencesI. 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