Send Orders for Reprints to [email protected] Current Tissue Engineering, 2014, 3, 119-129 119 The GFP Viability Assay for Cryobiology Applications: A Mechanistic Understanding Gloria D. Elliott1, John J. McGrath2 and Elahé Crockett3,* 1Dept. of Mechanical Engineering and Engineering Science, University of North Carolina at Charlotte, Charlotte, NC, US; 2Dept. of Aerospace & Mechanical Engineering, University of Arizona, Tucson, AZ, US; 3Dept. of Medicine, Michigan State University, East Lansing, MI, US Abstract: The authors have previously implemented Green Fluorescent Protein (GFP) transfection as a marker to assess viability both in vitro and in vivo following freezing injury, with loss of GFP fluorescence following treatment indicating cell death. Although excellent correlations with conventional vital dyes and staining methods (membrane integrity, histol- ogy) were observed following injurious freezing, until now the basis for the loss of GFP fluorescence was not comprehen- sively explored. In this work it was hypothesized that membrane breach caused by freezing causes leakage of GFP. Diffu- sion of GFP into the extra-cellular space then causes a loss of intracellular and average fluorescent signal as the GFP is di- luted and its fluorescent signal attenuated (diffusion-dilution hypothesis). A simple one-dimensional (1-D) mass diffusion equation implementing literature values of GFP diffusivity was found to adequately account for the observed time scale of GFP fluorescence attenuation in vitro. Conservation of mass was established by monitoring extracellular solution fluores- cence before and after cell lysis, which is consistent with the hypothesis of simple diffusion of a stable GFP molecule from the intracellular to extracellular space. The effect of freezing on the protein, external to the cellular environment, was investigated by repeated freezing of aqueous solutions of purified recombinant protein. A significant difference (p < 0.01) in fluorescence intensity between control samples and the frozen protein solutions was not observed until the third freeze- thaw cycle. These results suggest that cold denaturation of the protein is not a major contributor to GFP fluorescence loss following lethal freezing of cells and that the diffusion and dilution of the fluorophore is the basis of fluorescence loss. The intracellular GFP thus functions as a membrane integrity indicator following low temperature freezing injury. Keywords: Cell injury, fluorescent signal, freezing injury, gene transfer, GFP, protein. INTRODUCTION A number of studies have explored the use of GFP trans- fection as the basis for a viability assay following chemical The authors have previously shown that an assay based or physical stress, with each stress vector presenting unique on Green Fluorescent Protein (GFP) transfection can be used GFP signatures. For example, Baumstark-Khan et al. [14] to assess cell viability both in vitro and in vivo following assessed the colony forming ability of wild-type and GFP- freezing injury [1, 2]. GFP is a protein that is native to the transfected CHO cells after irradiation. They found that jellyfish species Aequorea victoria, in which it plays a role in colonies that survived radiation expressed GFP and that the light energy transfer [3]. The gene responsible for the pro- surviving fractions were comparable for transfected and duction of GFP was first cloned by Prasher et al. [4], and wild-type cells. They also analyzed irradiated cells using subsequently expressed in both prokaryotic and eukaryotic flow cytometry and found two distinct populations, cells that cells by Chalfie et al. [5]. Although GFP transfection was did not fluoresce green, and those that fluoresced with a first implemented as a tool for monitoring gene transfer [5, much brighter intensity. A hypothesis was formed that the 6], it has been applied widely since its introduction. Trans- non-fluorescing population suffered membrane damage and fection with GFP has been successfully implemented in tu- leaked GFP, while the brightly fluorescing population lost mor cell biology research, including metastasis and angio- the ability to divide but continued to produce protein. Leff genesis studies [7, 8]. It has been used to study cardiac de- and Leff [15] used GFP to measure the persistence of geneti- velopment and pathophysiology [9], virus transmission [10], 1 cally engineered E Coli upon introduction into different wa- bacterial pathogens , and aspects of basic cell physiology ter environments. They found that although cell counts based such as gap junctions [11]. Furthermore, GFP has also been on GFP were significantly less than when using traditional used as a marker of cell injury in laser-assisted cryosurgery staining and counting methods, the two reporting systems [12]. Efficient expression vectors have been developed for revealed similar trends with changing environmental condi- species as simple as the bacteria E. coli to more complex tions. Collins et al. [16] used GFP transfection of Mycobac- human mammalian cells [13]. terium tuberculosis as the basis for generating dose response *Address correspondence to this author at the Dept. of Medicine, Michigan curves for a range of anti-tuberculosis drugs, with compara- State University, East Lansing, MI, US; Tel: 517.432.8417; bility to traditional metabolic assays (correlation value = Fax: 517.432.9471; E-mail: [email protected] 0.89346). Still, others preferred the use of dual reporting 2211-5439/14 $58.00+.00 © 2014 Bentham Science Publishers 120 Current Tissue Engineering, 2014, Vol. 3, No. 2 Elliott et al. systems to unambiguously discern cell abundance and meta- cell injury. This concept will subsequently be referred to as bolic activity in bacterial samples [17], or counter-staining the dilution-diffusion hypothesis. with propidium iodide to distinguish living, compromised, and dead cells [18]. In previous work by the authors of the MATERIALS AND METHODS current article, loss of GFP fluorescence following low tem- perature exposure was explored as an indicator of cell death Cell Culture in tissues [19] and cell suspensions [2]. Although excellent GFP-transfected Rat 3230 Adenocarcinoma (R3230AC) correlations with conventional vital dyes and staining meth- cells were cultured in 50 ml Falcon flasks (Becton Dickin- ods (membrane integrity, histopathology) were observed son, Lincoln Park, NJ) containing Dulbecco’s modified Ea- following injurious freezing, the basis for the loss of GFP gle medium (DMEM) with 10% fetal calf serum and 4% fluorescence was hypothesized but not experimentally or penicillin-streptomycin solution (10,000 units/ml penicillin theoretically established. G sodium and 10,000 g/ml streptomycin in 0.85% saline). The continued development of GFP viability assays to- The cell line was a gift from Drs. Mark Dewhirst and Chuan gether with a mechanistic understanding of GFP fluores- Li (Duke University) and had been transfected in their labo- cence changes in the context of thermal injury can result in ratory with a peGFP-N1 plasmid (enhanced GFP) using its expanded utilization for a variety of injury modalities. For DMRIE liposomes and G418 selection. The enhanced GFP example, GFP was recently explored as a tool to assess he- (eGFP) is the red-shifted variant of wild-type GFP. All patic tissue viability following laser-assisted cryosurgery, products were purchased from Gibco BRL (Grand Island, which involves low and high temperature exposures [12]. NY). Cells were incubated at 37ºC in a 5% CO2 atmosphere. For lysis experiments, cells were grown to near confluence The GFP method has numerous advantages compared to 2 traditional viability assessment methods. An instantaneous, in 175 cm Corning flasks (Corning, NY). Cells were har- quantitative, non-invasive viability determination within vested by exposure to warm trypsin-EDTA solution (0.25%) living tissues can eliminate the need for labor-intensive, for 2 minutes. Cells were concentrated by centrifugation as time-consuming histological processing of tissues, and avoid necessary. the possibility of fixation artifacts. This method allows con- tinuous monitoring of spatio-temporal viability relationships Fluorescence Calibration Curve within living tissue and permit for monitoring of complex A calibration curve of fluorescencence intensity as a time-course experiments within a single animal rather than function of aqueous GFP concentration was constructed to using many animals euthanized at sequential times. This allow estimation of GFP concentration in the cell population. reduces the number of animals required for experimentation Calibration curves were constructed using recombinant en- and eliminates ambiguities that can be introduced by animal- hanced GFP, a red-shifted mutant of GFP (Clontech Labora- to-animal variations. Optically-based GFP tissue cell viabil- tories Inc., Palo Alto, CA). The GFP-transfected cells used in ity methods can be used together with other optical methods this study were transfected with the same red-shifted GFP (fluorescence-based or otherwise) for simultaneous meas- mutant. In thick liquid samples the fluorescent signal comes urements of physio-chemical, thermal, morphological, from both in-focus and out-of-focus regions of the sample physiological, inflammatory and immunological responses to with light absorption and scattering also influencing the freezing/thawing or other processes by using multi-spectral overall fluorescence measured [20]. To avoid possible arti- probes, infrared