1.

Introduction

Optical imaging is a powerful tool for monitoring the metabolic status of cells and tissues due to the auto-fluorescence properties of the metabolic co-enzymes NADH and FAD. NADH is the principal electron acceptor in glycolysis and electron donor in oxidative phosphorylation, while FAD is the primary electron acceptor in oxidative phosphorylation. The optical “redox ratio” (fluorescence intensity of NADH divided by that of FAD) is widely used to monitor cellular metabolism in cells, ex vivo tissues, and in vivo tissues in order to diagnose disease,^1,2 monitor cellular differentiation,³ and characterize other metabolic perturbations.^4,5

Recently, the fluorescence lifetimes of NADH and FAD have also been evaluated as optical metabolic biomarkers.^2,6–10 The fluorescence lifetime is the time a fluorophore remains in the excited state before returning to the ground state and emitting a fluorescent photon. Fluorescence lifetimes are self-referenced and more robust than fluorescence intensity measurements in regard to inter-system and intra-system variability.⁷ Additionally, fluorescence lifetimes are sensitive to changes in pH, temperature, and proximity to quenchers.^7,11 Often, the fluorescence lifetime decay curves for NADH and FAD are each fit to a two-component exponential decay.^2,6,12 The two lifetime components represent the two most probable configurations of NADH and FAD, which are free in solution or bound to a protein.^12,13 The short lifetimes and long lifetimes of NADH represent the free and protein-bound components, respectively. Conversely, the short and long lifetimes of FAD represent the protein-bound and free components, respectively. For these reasons, the fluorescence lifetimes of NADH and FAD are robust reporters for metabolic differences between tissues. Indeed, auto-fluorescence lifetime imaging has been used for monitoring pre-cancers and cancers in oral cancer models and in breast cancer models.^{2,6,10,14–18}

Ideally, optical metabolic measurements should be performed in vivo. Thus, these techniques have been predominantly used in easily accessible tissues, such as the skin and oral cancer models.^2,8,19,20 The growing development of endoscopes capable of confocal fluorescence microscopy is enabling measurements of previously inaccessible tissue.^21,22 However, fluorescence endoscopes are still somewhat specialized, cumbersome, and represent a significant technical and regulatory burden for feasibility studies, especially in tissue sites that are difficult to access (i.e., outside of the gastrointestinal tract). As a result, many feasibility studies in this field are performed on fixed tissue slides, immortalized cell lines, or excised tissue,^6,15,23 which may not accurately represent the metabolic state of live, intact tissue. Furthermore, the use of optical metabolic techniques would be greatly expanded by the development of an effective tissue handling protocol that allowed for ex vivo tissue interrogation that accurately reflects the in vivo metabolic state. However, the effect of ex vivo sample preparation on optical metabolic measurements has not been explored.

The goal of this study was to investigate live tissue culture as an alternative sample preparation for optical metabolic imaging when in vivo measurements are not feasible. Current protocols to represent in vivo metabolic activity in excised tissue require flash-freezing of the tissue;⁴ however, such a procedure requires tissue submersion in liquid nitrogen and may induce changes in sensitive fluorescence lifetime endpoints. Live tissue culture may address these limitations. Live tissue culture maintains the tissue in tissue culture media that provides oxygen, glucose, and other necessary nutrients. Furthermore, chilled tissue media has been used in previous optical studies^23,24 because it contains more dissolved oxygen than media maintained at body temperature. However, this chilled media procedure for maintaining tissue culture has not been rigorously compared to in vivo metabolic measurements.

We hypothesized that, for a discrete period of time following harvest, excised tissue maintained in chilled tissue culture media will provide metabolic measurements that are representative of the in vivo metabolic state. To test this hypothesis, the NADH and FAD fluorescence intensity and lifetime, as well as cellular morphology, of the cheek pouch epithelium of 16 Golden Syrian hamsters were measured in vivo, ex vivo from biopsies maintained in live culture over 48 h, and ex vivo from flash-frozen and thawed tissue biopsies. Both high and low time-resolution protocols were followed to investigate both short-term and long-term changes in the cellular metabolism of the cultured biopsies. For the high-resolution protocol, optical metabolic measurements were obtained every 30 min for 4 h from a single epithelial depth. For the low-resolution protocol, images of two different epithelial depths were acquired at 4, 8, 12, 24, and 48 h.

2.

Materials and Methods

3.

Results

Optical redox ratio values and NADH and FAD fluorescence lifetime values were acquired from the cheek pouch epithelium of 16 hamsters in vivo, from a cultured biopsy, and from a frozen-thawed biopsy. Figure 1 shows representative images of the normalized redox ratio, mean NADH lifetime, and mean FAD lifetime from the spinosum and basal layers of the epithelium in vivo, cultured tissue at 4, 12, 24, and 48 h after excision, and frozen-thawed tissue for the normal hamster epithelium. The redox ratio of the in vivo tissue appears to be similar to the cultured biopsy at 4 h and less than the cultured biopsy at 24 h. The frozen-thawed tissue appears to have a lower redox ratio than the in vivo tissue (Fig. 1). For the mean lifetimes of both NADH and FAD, the cultured tissue appears to have a lifetime similar to that of the in vivo tissue after 4 h; however, the frozen tissue shows an increased mean NADH lifetime relative to in vivo values (Fig. 1). No significant changes in cellular morphology are observed in the cultured or frozen tissue relative to the in vivo tissue measurements.

Fig. 1

Representative redox ratio (first row), NADH ${\tau }_m$ (second row), and FAD ${\tau }_m$ (third row) images from in vivo hamster epithelium at the basal (first column) and spinosum (second column) regions, tissue maintained in live culture 4, 12, 24, and 48 h after excision (third through sixth columns), and frozen-thawed tissue (last column). Each image is $100\times 100\mathit{\mu }\mathrm{m}$. ${\tau }_m$ is the mean lifetime (${\tau }_1*{\alpha }_1+{\tau }_2*{\alpha }_2$).

To compare deviations from the in vivo optical redox ratio in the cultured and frozen-thawed tissue samples, the optical redox ratio was normalized to the in vivo measurement. The variance in the redox ratio among in vivo measurements from the same hamster is 0.053, corresponding to a coefficient of variation (COV) of 8%. Figure 2 shows the normalized redox ratio of the in vivo tissue, cultured tissue over 4 h, and frozen-thawed tissue. No significant changes ($p>0.05$) in the redox ratio are observed at any time point for the 4 h the cultured biopsy was imaged [Fig. 2(a)]. The mean redox ratio of the cultured biopsy up to 4 h after excision is within 8% of the in vivo value. The redox ratio of the frozen-thawed sample, however, is 85% of the in vivo tissue measurement, and this difference is significant ($p<0.01$).

Fig. 2

(a) The redox ratio value of frozen-thawed (F) hamster epithelium is significantly reduced from the in vivo (IV) value, while live tissue culture maintains redox ratio values similar to in vivo measures over the 4 h of live culture measurement ($\text{mean}\pm \mathrm{SE}$ of $n=10$ measurements). (b) The redox ratios of the spinosum and basal epithelial layers of the live-culture biopsies over 48 h ($\text{mean}\pm \mathrm{SE}$ of $n=6$). Asterisk denotes significant difference between in vivo and the indicated measurement ($*p<0.05$; $**p<0.01$).

The metabolic activity of the stratified squamous epithelium is known to change with depth, with the most metabolically active cells occupying the basal layer of the epithelium.²⁸ Therefore, an analysis of the metabolic change in multiple epithelial layers was evaluated over time. The redox ratio of the basal region is greater than that of the spinosum region at all time points [$p<0.05$, Fig. 2(b)]. No significant change in the redox ratio is observed with time in live culture until 24 h, when the redox ratio of both the spinosum and basal cells increases [$p<0.05$, Fig. 2(b)]. By 48 h, the redox ratio in both regions decreases [$p<0.05$, Fig. 2(b)].

Quantitative parameters ($\text{mean}\pm \mathrm{SE}$) of NADH and FAD fluorescence lifetimes (${\tau }_m$, ${\tau }_1$, ${\tau }_2$, and ${\alpha }_1/{\alpha }_2$) for the in vivo images are presented in Table 1. The mean lifetime (${\tau }_m$) is a weighted average of the short (${\tau }_1$) and long lifetime (${\tau }_2$) components. Changes in hamster epithelium NADH fluorescence lifetime components relative to the in vivo values are shown in Fig. 3(a). Over the first 4 h, all of the mean NADH ${\tau }_m$ values from the live cultured biopsy are within 2.5% of the in vivo value ($p>0.05$), while the frozen-thawed NADH ${\tau }_m$ is $+13\%$ of the in vivo value, which is a statistically significant difference [$p<0.001$; Fig. 3(a)]. Similarly, for NADH ${\tau }_1$, all cultured biopsy measurements from the first 4 h are within 2% of the in vivo value ($p>0.05$), while the frozen-thawed ${\tau }_1$ is $+7\%$ [$p<0.01$; Fig. 3(a)]. For NADH ${\tau }_2$, the cultured biopsy values are within 1% of the in vivo NADH ${\tau }_2$ value ($p>0.05$) and the frozen-thawed mean ${\tau }_2$ is $+2\%$ [$p<0.05$; Fig. 3(a)]. The 4-h cultured biopsy NADH ${\alpha }_1/{\alpha }_2$ measurements are within 10% of the in vivo ${\alpha }_1/{\alpha }_2$ ratio ($p>0.05$), while the frozen-thawed biopsy mean ${\alpha }_1/{\alpha }_2$ is 81% of the in vivo value [$p<0.05$; Fig. 3(a)].

Fig. 3

Changes ($\text{mean}\pm \mathrm{SE}$ of $n=10$ measurements) relative to in vivo (IV) values of NADH (a) and FAD (b) ${\tau }_m$ (diamond), ${\tau }_1$ (square), ${\tau }_2$ (triangle), and ${\alpha }_1/{\alpha }_2$ (cross) observed in tissues maintained in live culture for 4 h, and in frozen-thawed (F) tissues. ${\tau }_m$ is mean lifetime (${\tau }_1*{\alpha }_1+{\tau }_2*{\alpha }_2$). Asterisk denotes significant difference between in vivo and indicated measurement ($*p<0.05$; $**p<0.01$; $***p<0.001$).

Table 1

Mean (SE) for in vivo measurements of NADH and FAD lifetime components (n=10).

Similar trends are observed in the FAD lifetime values [Fig. 3(b)]. Over the first 4 h, the mean FAD ${\tau }_m$ of the cultured biopsy increases slightly but stays within 8% of the in vivo FAD ${\tau }_m$ [$p>0.05$; Fig. 3(b)]. The frozen-thawed FAD ${\tau }_m$ is 9.5% greater than the in vivo FAD ${\tau }_m$ [$p<0.05$; Fig. 3(b)]. Changes in FAD ${\tau }_1$ are within 6% for the cultured biopsy measurement and frozen-thawed samples [$p>0.05$; Fig. 3(b)]. Likewise, the FAD ${\tau }_2$ values for the cultured and frozen-thawed samples vary less than 4% from the in vivo FAD ${\tau }_2$ [$p>0.05$; Fig. 3(b)]. While no significant difference is observed in the FAD ${\alpha }_1/{\alpha }_2$ ratio between the 4-h cultured biopsy and the in vivo FAD ${\alpha }_1/{\alpha }_2$ ($p>0.05$), the frozen-thawed measurement is 83% of the in vivo FAD ${\alpha }_1/{\alpha }_2$ ratio [$p<0.05$; Fig. 3(b)].

When the cultured biopsy is assessed past 4 h, significant changes in the NADH fluorescence lifetime components are observed (Fig. 4). Both the spinosum and basal layers follow the same trends. By 12 h, NADH ${\tau }_m$ significantly decreases by 10% and remains significantly lower than the in vivo value out to 48 h in both the spinosum and basal layers [$p<0.05$; Fig. 4(a)]. Again, at 12 h after excision, NADH ${\tau }_1$ and ${\tau }_2$ significantly decrease in both tissue layers [$p<0.05$; Fig. 4(b) and 4(c)]. NADH ${\alpha }_1/{\alpha }_2$ is increased from the in vivo measurement at 12, 24, and 48 h after excision [$p<0.05$; Fig. 4(d)].

Fig. 4

Changes ($\text{mean}\pm \mathrm{SE}$ of $n=6$ measurements) relative to in vivo (IV) values of NADH ${\tau }_m$ (a), ${\tau }_1$ (b), ${\tau }_2$ (c), and ${\alpha }_1/{\alpha }_2$ (d) observed from both the spinosum (diamond) and basal (square) epithelial layers of tissues maintained in live culture for 48 h. ${\tau }_m$ is mean lifetime (${\tau }_1*{\alpha }_1+{\tau }_2*{\alpha }_2$). Asterisk denotes significant difference between in vivo and indicated measurement ($*p<0.05$; $**p<0.01$).

Changes in the FAD lifetime of the spinosum layer are observed in the cultured biopsy at 48 h, when the FAD ${\tau }_m$ is significantly increased over the in vivo value [$p<0.01$; Fig. 5(a)]. Both FAD ${\tau }_1$ ($p<0.01$) and ${\tau }_2$ ($p<0.01$) of the spinosum also increase at 48 h [Fig. 5(b) and 5(c)]. FAD ${\alpha }_1/{\alpha }_2$ decreased at 48 h in both the spinosum ($p<0.05$) and basal cells [$p<0.05$; Fig. 5(d)].

Fig. 5

Changes ($\text{mean}\pm \mathrm{SE}$ of $n=6$ measurements) relative to in vivo (IV) values of FAD ${\tau }_m$ (a), ${\tau }_1$ (b), ${\tau }_2$ (c), and ${\alpha }_1/{\alpha }_2$ (d) observed from both the spinosum (diamond) and basal (square) epithelial layers of tissues maintained in live culture for 48 h. ${\tau }_m$ is mean lifetime (${\tau }_1*{\alpha }_1+{\tau }_2*{\alpha }_2$). Asterisk denotes significant difference between in vivo and indicated measurement ($*p<0.05$, $**p<0.01$).

Cellular morphology is maintained across the tissues regardless of preservation method (Fig. 6). No significant change ($p>0.05$) is measured in the average cellular cross-sectional area between the in vivo tissue, cultured biopsy, and frozen biopsy [Fig. 6(a) and 6(b)]. Likewise, the NCR remains unchanged ($p>0.05$) among the in vivo tissue, cultured biopsy, and frozen biopsy [Fig. 6(c) and 6(d)].

Fig. 6

Cellular morphology, as measured by cell cross-sectional area [(a) and (b)] and nuclear-to-cytoplasmic ratio [(c) and (d)] remains unchanged ($p>0.05$) from in vivo (IV) values within both the spinosum and basal epithelial layers of tissues maintained in live culture over 48 h and in frozen-thawed (F) tissues ($\text{mean}\pm \mathrm{SE}$ of $n=10$ [$n=6$ for (b) and (d)] measurements of a minimum of 20 cells per image).

Histological analysis further confirms that the live tissue culture method maintains tissue viability. H&E stains reveal similar structure in the epithelial layer of the tissue at 4, 12, 24, and 48 h after excision (Fig. 7). Positive staining with antibodies against Ki67 reveals proliferating cells in the basal layer at all time points (Fig. 7). Furthermore, no apoptotic cells are observed in cleaved-caspase 3 stains of the epithelium at any time point (Fig. 7). Together, the histological analysis suggests that the cells of the hamster epithelium remain alive in tissue culture for 48 h after excision.

Fig. 7

Representative histology of the hamster epithelium. Hematoxylin and eosin staining demonstrates an intact epithelium (first row). Nuclear Ki67 staining (brown) of the basal cells suggests cellular proliferation at each time point (middle row). An absence of staining by anti-cleaved caspase-3 suggests no apoptosis (third row). Images were acquired using a $40\times$ air objective.

4.

Discussion

The goal of this study is to determine whether optical metabolic imaging of ex vivo tissues maintained in live culture reflects in vivo measurements. Optical endpoints include the optical redox ratio, NADH fluorescence lifetimes, and FAD fluorescence lifetimes, which are measured from in vivo tissue, a biopsy maintained in chilled tissue media, and a flash-frozen and thawed biopsy. Minimal changes, within 10% for the redox ratio and within 8% for the lifetime values, were observed between the in vivo hamster epithelium and the cultured tissue through 8 h after excision. However, statistically significant changes were observed in the frozen-thawed tissue. These results suggest that optical metabolism measurements from cultured tissue within 8 h after excision represent the in vivo metabolic state of the tissue, especially when compared to frozen-thawed tissue.

Live tissue culture is used in many applications, including optical metabolic imaging and biochemical metabolic studies, to maintain viability of excised tissue.^23,29–31 However, the temporal impact of ex vivo culture on the metabolic state of tissues has not been quantitatively assessed previously. Often, studies use the tissue for several days³⁰ rather than the few hours investigated here. Cell viability studies have shown that for epithelial tissue, less than 2% cell death occurs over the first 5 days.³⁰ However, no previous studies have confirmed that this live tissue culture approach provides optical metabolic measurements that are consistent with in vivo values, as we have shown in the current study. This live tissue culture approach is particularly attractive for optical imaging because these methods interrogate superficial layers of tissue ($\sim 200\mathit{\mu }\mathrm{m}$ for two-photon microscopy, depending on tissue type), so that oxygen and nutrients can diffuse into the tissue and maintain viability.^32,33 These superficial interrogation volumes also avoid the necrotic and hypoxic cores that are found in some live tissue culture preparations.³⁴

The optical redox ratio has been previously used to quantify metabolic differences among normal tissue, pre-cancerous tissue, and cancerous tumors.^1,2,26,35,36 In the current study, two epithelial depth layers were interrogated because stratified squamous epithelial tissues are known to have a gradient of cellular metabolic activity, with the basal cells being most active and the superficial cells being least active.^2,28 As shown in Fig. 2(b), the redox ratio of the basal region is greater ($p<0.05$) than the spinous region in vivo and at every time point during the live culture experiment, as expected. Additionally, Fig. 2(a) suggests that the optical redox ratio of frozen-thawed tissue does not completely describe the in vivo metabolic state. While the optical redox ratio is significantly reduced in frozen-thawed samples ($p<0.01$), no significant difference is observed in the cultured tissue until 24 h after biopsy [Fig. 2(b)]. The change in redox ratio in the frozen-thawed tissue is due to both a decrease in the NADH fluorescence intensity and an increase in FAD fluorescence intensity (data not shown), which is consistent with decreased NADH fluorescence intensity observed between fresh and frozen-thawed hamster and cervical tissues.^25,31 The NADH and FAD fluorescence intensities did not change significantly in the live tissue culture over 4 h ($p>0.05$).

The mean NADH lifetime of the hamster cheek epithelium is 1.149 ns (Table 1), which is consistent with previously reported NADH lifetime values.^2,6,10,37,38 The mean NADH fluorescence lifetime is the averaged effect of the free and protein-bound lifetime components.^2,11 While only minimal changes are observed in the NADH lifetime components of the cultured tissue until 12 h after biopsy, the frozen-thawed tissue has an increased ${\tau }_m$, ${\tau }_1$, and ${\tau }_2$ and a decreased ${\alpha }_1/{\alpha }_2$ ratio (Figs. 3 and 4). These results indicate that the fluorescence lifetimes of both the bound and free NADH are increased in the frozen-thawed tissue and that a greater portion of NADH exists in the bound conformation. Cells undergoing stress, such as apoptosis, have also shown increased mean NADH fluorescence lifetimes and increased proportions of bound NADH, suggesting that the freeze-thaw process exerts more stress than the live culture technique.³⁷

Likewise, the observed FAD mean lifetime of 0.829 ns (Table 1) is similar to reported FAD lifetime measurements.^2,39 For the FAD lifetime components, the frozen-thawed tissue has an increased ${\tau }_m$ and a decreased ${\alpha }_1/{\alpha }_2$ (Fig. 3). The decreased ${\alpha }_1/{\alpha }_2$ ratio indicates an increase in the relative amount of free (long-lifetime) FAD. This shift in relative amounts of the free and bound lifetime contributions explains the increased FAD ${\tau }_m$. No significant change is observed in the FAD ${\tau }_1$ and ${\tau }_2$, so the FAD fluorescence lifetime may be less sensitive to the cellular stress involved in the freeze-thaw process than the NADH fluorescence lifetime.

Consistent cellular morphology across the in vivo, cultured, and frozen-thawed tissue indicates maintenance of tissue structure. The average cell cross-sectional areas observed in the hamster epithelial spinosum and basal layers are 530 and $200{\mathit{\mu }\mathrm{m}}^2$, respectively [Fig. 6(a) and 6(b)], which are consistent with published cell cross-sectional areas of hamster epithelium.⁴⁰ Likewise, the observed NCR of the spinosum and basal layers of 0.2 and 0.4 [Fig. 6(c) and 6(d)], respectively, is consistent with previous reports.^40,41 The current study is also consistent with previous studies reporting that cell size and NCR do not change in frozen-thawed cells/tissues or in live tissue culture relative to in vivo values.^41,42

While the optical metabolic measurements varied from the in vivo values after 8 h in live culture, cellular morphology and histological analysis revealed no changes in cellular morphology, proliferation, or cell death over the 48-h time-course (Figs. 6 and 7). Therefore, optical metabolic imaging endpoints may be more sensitive to subcellular molecular changes than the histological or morphological analyses. Previous studies of the live tissue culture method have used histology to verify cell viability;^30,33 however, our results suggest that molecular metabolic changes occur before changes in cellular proliferation or cell death. Thus, optical metabolic imaging may be a more sensitive endpoint than histology for detecting changes in cell status.

To validate live tissue culture as an effective protocol for maintaining in vivo metabolic characteristics in excised tissue, the optical redox ratio, NADH fluorescence lifetime, and FAD fluorescence lifetime were quantified from hamster cheek epithelia in vivo, in live cultured biopsies followed for 48 h, and in frozen-thawed samples. The live tissue culture approach resulted in no significant change in any optical endpoint relative to in vivo measures until 12 h, when a decreased NADH ${\tau }_m$ was observed. Several significant differences, including a reduced redox ratio, increased NADH ${\tau }_m$, and increased FAD ${\tau }_m$ were observed in the frozen-thawed samples. These results indicate that the live tissue culture method represents the in vivo state more accurately than the frozen-thawed procedure. Therefore, when in vivo optical measurements are not feasible, excised tissue may be maintained in chilled tissue media up to 12 h for close representation of in vivo metabolic states.