Buccal mucosa in vitro experiments: I. Confocal imaging of vital staining and MTT assays for the determination of tissue viability

Journal of Controlled Release 58 (1999) 39–50

Buccal mucosa in vitro experimentsI. Confocal imaging of vital staining and MTT assays for the

determination of tissue viability1 ,*Delphine Imbert , Christopher Cullander

Department of Biopharmaceutical Sciences, School of Pharmacy, University of California at San Francisco, 513 Parnassus Avenue,Box 0446, San Francisco, CA 94143-0446, USA

Received 7 April 1998; received in revised form 10 July 1998; accepted 29 July 1998

Abstract

Delivery of drugs through the skin and the buccal mucosa has been considered as an alternative to per oral dosing forthose substances that are degraded in the gastro-intestinal tract, or are subject to first-pass metabolism in the liver. In thebuccal mucosa, contrary to skin, the diffusion barriers are located within living cell layers, hence the physiological state ofthe tissue is likely to significantly affect in vitro diffusion profiles. In this study, we were interested in assessing the viabilityof excised buccal mucosa and determining the limits of tissue usage under common in vitro experimental conditions. Usingconfocal laser scanning microscopy (CLSM), we have shown that optical sectioning of samples exposed to calcein AM andethidium homodimer-1 (used as ‘live’ and ‘dead’ cell probes respectively) can be employed to accurately and reliablydetermine the viability of buccal mucosa biopsies. The results of the CLSM assay were remarkably consistent with that of anMTT assay. In both studies, viability in PBS at 348C was lost after about 8 h post-mortem, whereas it could be sustained forup to 24 h in KBR. 1999 Elsevier Science B.V. All rights reserved.

Keywords: Buccal mucosa; Viability; Confocal microscopy; MTT assay; Vital staining

1. Introduction liver [1–3]. In transdermal delivery, the rate-limitingbarrier for most compounds resides within the

Delivery of drugs through the skin and the buccal stratum corneum, thus, viability has been ignored inmucosa has been considered as an alternative to per most in vitro skin studies unless effects of metabo-oral dosing for those substances (e.g., peptides and lism were specifically addressed [4]. In the buccalproteins) that are degraded in the gastro-intestinal mucosa, however, the diffusion barriers are locatedtract, or are subject to first-pass metabolism in the within living cell layers, hence the physiological

state of the tissue is likely to affect diffusion profilesobtained in vitro to a much greater extent than when

*Corresponding author. Tel.: 11-650-802-1877; fax: 11-650- working with skin or other keratinized epithelia [5].595-2580; email: [email protected]

1 In addition to obtaining reliable permeabilityCurrent address: Cellegy Pharmaceuticals, Inc., 349 Oystercoefficient, the use of viable tissue in diffusionPoint Boulevard, Suite 200, South San Francisco, CA, 94080,

USA. experiments also opens up the scope of possibilities

0168-3659/99/$ – see front matter 1999 Elsevier Science B.V. All rights reserved.PI I : S0168-3659( 98 )00143-6

40 D. Imbert, C. Cullander / Journal of Controlled Release 58 (1999) 39 –50

for in vitro screening. Although more permeable than 2,5-diphenyltetrazolium bromide) and dimethylsul-skin, buccal mucosa still represents an effective foxide (DMSO) were obtained from Sigma (Saintbarrier to most xenobiotics and permeation enhance- Louis, MO). Krebs Ringer bicarbonate (KBR) bufferment strategies are required to provide therapeutic was prepared within 24 h of use with 115.5 mMdrug blood levels. One such approach—prodrug NaCl, 4.2 mM KCl, 2.5 mM CaCl , 1.6 mM2

design—, for example, can only be evaluated in vitro NaH PO , 0.8 mM MgSO , 4.0 mM HEPES, 17.32 4 4

with viable tissue. The use of viable tissue also mM Na CO , and 12.2 mM glucose. Buffer pH was2 3

allows a simultaneous in vitro evaluation of chemical brought down to around 8.0 with dry ice andpermeation enhancers for both efficacy and toxicity equilibrated to 7.5 with carbogen (95% O215%[6]. Because it has not been well documented in the CO2) bubbling for about one h. After overnightliterature, we were interested in assessing the viabili- storage at 48C, KBR was equilibrated to roomty of excised pig buccal mucosa and determining the temperature and its pH was adjusted before uselimits of tissue usage under typical in vitro ex- (when necessary) by carbogen bubbling. All otherperimental conditions. chemicals were of analytical grade.

We examined viability using two assays which hadnot been developed for buccal mucosa before: (1) 2.2. Animalsconfocal laser scanning microscopy (CLSM) imag-ing of the vital stains calcein AM (CAM) and Pig buccal mucosa was obtained from the UCSFethidium homodimer-1 (EH-1), and (2) MTT assay. Experimental Surgery Department (San Francisco,The optical slicing capability of CLSM permits CA) through the UCSF Tissue Sharing Program. Pigsvisualization of cells deep in thick tissue samples (females, 80–120 pounds) were sacrificed in thelike buccal mucosa biopsies which are sufficiently course of other studies. Both cheeks were isolatedtransparent, do not scatter light strongly, and are not immediately using disposable razor blades and im-strongly autofluorescent. The combination of CAM mersed in phosphate-buffered saline (PBS) pH 7.4 or(a non-fluorescent cell-permeant dye which is Krebs Ringer bicarbonate buffer (KBR) pH 7.5 atcleaved by intracellular esterases to fluorescent cal- room temperature, as indicated in text. In all studies,cein) and EH-1 (which passes through damaged cell euthanasia and tissue excision occured within 5 min.membranes to bind DNA and undergo a 40-fold Less than 15 min post-mortem, skin and connectiveenhancement in fluorescence) has proven useful for tissues were rapidly removed with a disposable razorviability and cytotoxicity determination in monolayer blade. The mucosa was then either dermatomedcultures [7]. It has been used successfully in our using a Padgett Electro Dermatome (Padgett Derma-laboratory to assess the effect of storage on the tome Division, Kansas City Assemblage Co., Kansasviability of both epithelium and endothelium in City, MO) set at 800 mm, or carefully dissectedexcised corneas [8]. using surgical scissors. Sheets of mucosa were

Similarly, the MTT assay is a well-established floated for up to 30 h onto various media, includingviability and cytotoxicity assay in cell cultures [9], PBS pH 7.4, PBS pH 7.4 with 0.02% NaN , KBR3

which has been adapted for the determination of skin pH 7.5, KBR pH 7.5 with 0.02% NaN or KBR pH3

sample viability [10,11] and, recently, in our labora- 7.5 without glucose. Viability experiments weretory for that of excised cornea [8]. conducted at room temperature, at 348C, or at 378C,

as indicated in the text. In all graphs, the origin ofthe time axis refers to the time post-mortem (65

2. Materials and methods min), not to the time when the experiment wasstarted.

2.1. Chemicals and reagents2.3. Viability assays

Ethidium homodimer-1 (EH-1) and calcein AM(CAM, the acetyloxymethyl ester of calcein) powder 2.3.1. MTT Assaywere purchased from Molecular Probes, Inc. MTT is a yellow, water-soluble compound that is(Eugene, OR). MTT (3-[4,5-dimethylthiazol-2-yl]- enzymatically reduced to dark purple and insoluble

D. Imbert, C. Cullander / Journal of Controlled Release 58 (1999) 39 –50 41

formazan by viable cells. The MTT assay protocol tetrazolium salt that is reduced to a soluble ratherused in this study was adapted from earlier studies than insoluble formazan), which can be reducedconducted in our laboratory with excised cornea [8]. under certain conditions without cells or enzymes.

The day of the assay, 2 mg/ml MTT was dis- We also verified that MTT was chemically stablesolved in freshly-prepared PBS. Any undissolved under our experimental protocol.crystals were removed by filtration through aNalgene 0.45-mm nylon syringe filter (Nalge Co., 2.3.2. CLSM assayRochester, NY). Each experiment was conducted Four-millimeter biopsy samples were obtainedusing tissue from a single animal and three (up to from dermatomed buccal sheets floated on media atsix) replicates were obtained for each data points. time points up to 27 h post-mortem and rinsed withFour-millimeter biopsy samples were obtained from the appropriate buffer. Each biopsy was immersed inthe buccal tissue sheets using a disposable biopsy 150 ml of PBS pH 7.4 or KBR pH 7.5 containing 25punch (Baker-Cummins Dermatologicals, Inc., mM EH-1 and 50 mM CAM prepared immediatelyLakewood, NJ) at time points up to 27 h post- before use from a 2 mM EH-1 stock solution in 4:1mortem and then rinsed with the appropriate buffer. water /DMSO and a 10 mM CAM stock solution inExcess solution was carefully removed and the anhydrous DMSO. After staining (150 min), thebiopsies were weighed and placed into individual sample was rinsed with buffer and positioned (with-wells of a six-well tissue culture plate (Becton out any processing or fixation) in a sample holderDickinson, Lincoln Park, NJ). Two milliliters of designed in our laboratory so that the anatomicalMTT solution were added to each well, and the plate surface of the tissue was orthogonal to the coverslip.was incubated for 2 h at 378C on a rotating platform To help support the biopsy, cylinder-shaped surgical(250 rpm). After incubation, the remaining MTT sponges with cross-sections equal to that of thesolution was removed by aspiration, and the tissue biopsy (4 mm) were used.was rinsed twice with 1 ml of PBS for 1 min. Tissue The confocal microscope system used in this studywas then minced with surgical scissors, and the was a BioRad MRC600 (BioRad, Hercules, CA)formazan precipitate was extracted in 4 ml of equipped with a Kr /Ar laser and mounted on aDMSO, agitating with a tilted, rotating platform at Nikon Optiphot microscope. Samples were simul-80 rpm for 80 min. Formazan absorbance was taneously excited with the 488-and the 568-nm linesmeasured at 540 nm with DMSO as a blank. and imaged with Nikon dry (CF Fluor 103 /0.5) orFormazan production has been linked to several oil-immersion (CF Fluor DL 403 /1.3 and CF Nenzymes [12]. For the purpose of this work, the Plan Apochromat DM 603 /1.40) lenses and a zoomgroup of enzymes responsible for the reduction of of 1. Calcein and EH-1 fluorescence were detectedMTT was referred to collectively as tetrazolium with the BioRad K1/K2 filterblock set. It should bereductase (TR) and data were expressed in absor- noted that in samples stained with both probes andbance units per mg tissue (TR index). Results were scanned simultaneously with the 488- and the 568-analyzed by one-way ANOVA and, when necessary, nm (as in this study), a significant bleedthrough ofnon-parametric Scheffe post-test using StatView 4.1 the calcein signal was visible in the EH-1 channel.(Cherwell Scientific Publishing, Inc., Palo Alto, CA). Such bleedthrough could be easily avoided by turn-Differences in values were reported as significant ing off the 488-nm line when acquiring EH-1when P,0.05. The first assay (control) was typically images. However, for the sake of simplicity andbegun within 30 min post-mortem. Control TR because the two probes had different staining pat-indices were dependent upon (1) the time elapsed terns and each could be easily discerned, all confocalbetween the tissue excision and the start of the assay images in this study were acquired with simultaneousand (2) the nature of the transfer buffer. Both are scanning of the 488- and the 568-nm lines.provided in Section 3. While concerns have been Optical sections were obtained in planes parallel toraised about the stability and sensitivity of tetra- the cover slip (i.e., orthogonal to the anatomicalzolium salts in viability assays [12], MTT reduction surface of the sample) well below any cells damagedhas been shown to be dependent upon enzymes of by the biopsy punch (Fig. 1). As described earlierthe endoplasmic reticulum, unlike that of XTT (a [13,14] and as illustrated on Fig. 1, this orientation


Fig. 1. CLSM imaging of buccal mucosa biopsies. The tissue sample is positioned (without any processing or fixation) with the anatomicalsurface of the tissue perpendicular to the coverslip (not represented). Optical sections are obtained in planes perpendicular to the anatomicalsurface of the sample, well below any cells damaged by the biopsy punch. Such orientation of the tissue sample allows fluorescence imagingof all layers of the epithelium in the same optical plane.

of the tissue sample allows the imaging of all cell deep enough into the biopsy to ensure that nolayers of the epithelium in the same optical plane damage from the biopsy punch was visible; yet, it(i.e., at the same depth below the coverslip). In was close enough to the coverslip for good intensitypreliminary experiments, the optimal imaging depth and resolution. Typically, using the Nikon CF Fluorwas determined using viable samples double-stained DL 403 /1.3 lens, we imaged at a depth of about 40with EH-1 and CAM. In the area of the tissue mm, while with the Nikon CF Fluor 103 /0.5 (whichimmediately in contact with the cover slip (i.e., gave a much thicker optical section), imaging couldtissue that was sectioned by the biopsy punch), high performed at a depth of about 70 mm.levels of cell death could be observed on the EH-1 EH-1 images were analyzed using the particlechannel, and no calcein signal was detected. As the counting capability of NIH image V. 1.60 and theimaging plane was moved downward and viable cells downloadable confocal macros. Briefly, EH-1 imageswere reached, the EH-1 signal decreased dramatical- were opened in NIH Image with a red density slicely, and the calcein signal increased. As one pro- set so that all EH-1-stained nuclei appeared in red.ceeded further down, the calcein signal started Outlining, labeling and counting of the color-codeddecreasing, either because CAM might not have nuclei was then performed automatically using thediffused that far, or simply because of attenuation Analyze Particles function of the Analyze menu. Thefrom intervening cell layers. All our CLSM imaging area analyzed was also measured and results were

2was performed in a plane positioned about 10 mm expressed as number of EH-1 stained nuclei per mmbelow the plane of brightest calcein signal. This was of tissue imaged.


3. Results and discussion

3.1. MTT assay

A significant decrease in TR index was observedwith dissected buccal mucosa stored in PBS at roomtemperature as early as 3 h post-mortem (Fig. 2,open symbols). The influence of temperature onviability was studied in a separate experiment, usingtissue from a second animal (solid symbols). Asshown on Fig. 2, TR indices at 378C were sig-nificantly lower than those at room temperature(218C628C) at all time points. Additionally, theclose similarity between the two sets of data obtained Fig. 3. MTT assay of dermatomed buccal mucosa: Influence of

medium composition at room temperature. Tissue was excisedat room temperature from two different animalsfrom the animal immediately after sacrifice and transfered to theillustrate the robustness and good reproducibility oflaboratory in KBR at room temperature. Buccal mucosa was

the assay. dermatomed to 800 mm, stored in various media at roomAlthough PBS is still used by some groups as a temperature, and assayed 3 h and 20 h post-mortem (Mean6s.e.,

flow-through buffer [15], most now use KBR-type n53).

buffers in an effort to sustain the viability of themembrane. We wanted to evaluate whether theviability of dermatomed mucosa was indeed im-proved by such buffer systems. Moreover, because after 2.5 h in PBS and in KBR is significant,KBR buffers contain glucose, and due to the high although not as important as we expected. After 20bacterial load of the buccal mucosa, the influence of h, however, PBS yielded TR index 50% lower thansodium azide was also investigated. Fig. 3 shows those obtained from KBR. Additionally, no signifi-that, at room temperature, the difference in viability cant differences were found between PBS and KBR

lacking glucose, or when comparing media with orwithout sodium azide. In their MTT assay with skin,Hood et al. used a 50% decrease from control as alimit for viable samples [11]. Control samples inKBR assayed within 1 h post-mortem reproduciblyyielded TR indices around 0.05 (Figs. 3 and 4).Thus, by Hood’s criteria (TR indices above 0.025 arefrom viable samples), after 20 h in KBR (with orwithout sodium azide) at room temperature, buccalsamples were still viable (TR indices of 0.035 and0.038, respectively) whereas those in PBS or in KBRwithout glucose were not (TR indices of 0.020 and0.021 respectively).

Because most in vitro diffusion studies are con-ducted at 348C, a study similar to that in Fig. 3 wasconducted at that temperature. TR indices for KBR

Fig. 2. MTT assay of dissected buccal mucosa: Influence of samples were maintained at 85% of the control valuetemperature. Tissue was excised from the animal immediately after 6.3 h and at 72% after 9.3 h (Fig. 4a). Contraryafter sacrifice and transfered to the laboratory in PBS at room

to Fig. 3, however, striking differences in TR indicestemperature. Buccal mucosa was carefully dissected using surgicalbetween KBR and PBS samples were visible at allscissors, floated on PBS at room temperature and assayed at

various time points post-mortem (Mean6s.e., n53–5). time points. As early as 3.3 h post-mortem, TR


(Fig. 6). In PBS, there was a significant increase innuclear staining with time for about 9 h post-mor-tem. In some cases, this increase was highly linear

2(Fig. 6, closed circles, R 50.90); in others, however,the trend was not as clearly marked, particularly athigher temperatures (Fig. 6, open squares). Neverthe-less, in all cases, about 9 h post-mortem, the numberof EH-1 nuclei in the KBR samples was significantlylower (|50%) than that in the PBS samples, which isremarkably consistent with the MTT assay. After 10h in PBS, loss of tissue structure could be observedby CLSM, and translated into an apparent decreaseof the number of EH-1 nuclei. Despite these en-couraging results, however, the EH-1 signal acquisi-tion and quantification was tedious and often pro-vided noisy results at early time points. Therefore,we concentrated our efforts on the calcein results.

CLSM images of the calcein fluorescence detectedat the basal level of buccal mucosa samples stored at348C in PBS or in KBR are shown in, respectively,Figs. 7 and 8. After about 9 h post-mortem, nocalcein fluorescence could be elicited from the PBSsamples as illustrated on Fig. 7d. In KBR samples,however, calcein fluorescence was detected morethan 24 h post-mortem (Fig. 8d).

3.3. DiscussionFig. 4. MTT assay of dermatomed buccal mucosa: Influence ofmedium composition at 348C. Tissue was excised from the animal

Viability is a parameter that is gaining increasedimmediately after sacrifice and transfered to the laboratory inKBR (a) or PBS (b) at room temperature. Buccal mucosa was attention in studies where freshly excised membranesdermatomed to 800 mm (a) or dissected (b), stored in various are used to mimic in vivo conditions, as in in vitromedia at 348C, and assayed at various time points post-mortem

diffusion experiments. With skin, viability may not(Mean6s.e., n53).critically affect in vitro diffusion behavior, providedthat the stratum corneum is the main barrier to

indices from PBS samples were down to 0.03 (i.e., diffusion, an hypothesis which holds true in many37% decrease from the KBR control). A 50% circumstances. However, in instances where metabo-decrease was reached at 6.3 h. These results were lism is of interest, where the compound’s diffusion isconfirmed in a separate experiment (Fig. 4b). epidermis-limited, and where non-keratinized epi-

thelia (like buccal mucosa and cornea) are studied,3.2. CLSM assay viability should be carefully evaluated as it has the

potential to directly affect results obtained in vitro.In the CLSM assay, EH-1 images of buccal Numerous viability assays have been developed

mucosa kept at room temperature in PBS showed an for a large array of cell lines and cytotoxic events,51 3increase in nuclear staining with time (Fig. 5). Using including Cr release, H-thymidine uptake [16],

standard image analysis techniques, the number of trypan blue exclusion, ATP and glucose utilization,EH-1 stained nuclei per unit surface area imaged was tetrazolium salts colorimetric assays [16,17] andquantified and plotted as a function of time post- fluorescent vital staining.mortem for tissue samples stored in PBS or KBR Only a limited number of these assays, however,


Fig. 5. CLSM imaging of EH-1 fluorescence in the upper layers of the buccal mucosa at 2.5 h (left) and 7.0 h (right) post-mortem. Scale barrepresents 100 mm.

have been or can be adapted to the study of thick [4,22,23] are two popular skin viability assays.tissue sample viability. The tetrazolium salts assays Thymidine uptake [24] and trypan blue exclusion[10,11,18–21] and the glucose utilization assays [25,26] were used recently with cornea. Ferrera et al.

determined heart biopsies viability using the MTTbioassay [20]. Recently, Potter and coworkers [27]used nuclear fluorescence staining to estimate in vivotissue injury following laser surgery. However,fluorescent viability probes, although widely used incell cultures, have only found a limited number ofapplication with thick samples [28].

Typically, buccal tissue viability has been assessedthrough a combination of ATP measurements [29]and histopathological examinations [30], com-plemented (or not) by linearity of transport data[30,31]. One could argue that the latter is a test ofintegrity rather than viability, as are electrophysio-logical measurements (transepithelial potential andresistance), which are also commonly used not onlyfor buccal mucosa [32–34], but for other epithelia as

Fig. 6. Quantitative assessment of viability by CLSM. Surfacewell [33,35]. We have addressed this point in detaildensity of nuclei stained by EH-1 as a function of time post-elsewhere [36,37]. Nevertheless, despite this aware-mortem at the end of staining. Buccal mucosa was dermatomed to

800 mm and stored in PBS at room temperature (d), PBS at 348C ness, only limited viability data are published in(h), or KBR at 348C (s). Each experiment was conducted these studies. This is of concern with respect to theseparately using tissue from a different animal. One biopsy was interpretation of flux data when most laboratoriesimaged throughout the thickness of the epithelium at each time

using porcine buccal mucosa get their tissue frompoints, at 2–4 different locations. Straight lines indicate regression2 slaughterhouses, and the time of death (often un-lines for PBS at room temperature (d, 2–9 h, R 50.90) and KBR

2at 348C (s, 2–11 h, R 50.27). known or rarely reported) seldom coincides with the


Fig. 7. CLSM imaging of calcein fluorescence in the basal layers of dermatomed buccal mucosa stored in PBS at 348C, 2.8 h (a), 4.8 h (b),6.8 h (c), and 8.8 h post-mortem. Scale bar represents 100 mm.

time of tissue excision. Furthermore, once excised, and the time the tissue is actually excised by thealthough it is a common practice to keep buccal researchers. The decrease in viability is sharper astissue in KBR or PBS (ice-cold during transfer from the temperature is increased to 348C (Figs. 3 and 4)the slaughterhouses, and at 348C during diffusion and 378C (Fig. 2). We have not investigated the rateexperiments), limited data are available to document of TR decrease at 48C, however it is likely to bethe extent to which viability is actually maintained slower than at the above temperatures. This wouldwith these media [38]. In our laboratory, the MTT constitute relevant information, however, for lab-assay and CLSM vital staining with the EH-1/CAM oratories obtaining tissue from carcasses storedprobe pair were used earlier to assess the viability of overnight in cold rooms [39].excised rabbit corneas. In this study, we adapted Having established that the MTT assay can suc-these two assays for the study of buccal mucosa cessfully discriminate between various experimentalviability. parameters (time and temperature) and shows a good

Contrary to the common belief that buccal mucosa inter-animal reproducibility, buccal mucosa viabilityviability is relatively stable for about 6 h [32], we was then determined under conditions closer to thosehave shown that TR activity in dissected mucosa used in in vitro experimental setups. With a fewdecreases sharply in the first 10 h post-mortem at exceptions [15,40], most in vitro buccal studies areroom temperature (Fig. 2). These experimental con- conducted using dermatomed mucosa with KBRditions are close to those encountered in the slaug- rather than PBS as diffusion cell medium [41,33]. Athterhouse between the time the animals are sacrificed room temperature, the difference between KBR and


Fig. 8. CLSM imaging of calcein fluorescence in the basal layers of dermatomed buccal mucosa stored in KBR at 348C, 2.7 h (a), 8.7 h (b),10.7 h (c), and 25 h post-mortem. Scale bar represents 100 mm.

PBS was statistically significant but practically mini- consistently affect viability compared to the originalmal (Fig. 3). At 348C, however, the temperature of media. Glucose however, was shown to be indispens-choice for in vitro diffusion studies, differences able to the maintenance of TR activity in the tissuebetween the two media were dramatically accen- (Fig. 3).tuated. As early as 3.3 h post-mortem, PBS samples These MTT results were remarkably consistentyielded TR indices 60% lower than those from KBR with both the EH-1 and calcein results obtained bysamples (Fig. 4b), whereas buccal mucosa viability CLSM. Obviously, as opposed to the MTT assay,in KBR was maintained above 72% of its original CLSM imaging cannot be used as a routine labora-value for more than 9 h post-mortem (Fig. 4a). The tory assay for viability determination and screening.influence of sodium azide (NaN ), an antimicrobial Furthermore, as we showed in this study, CLSM3

commonly used in in vitro skin studies, was also does not readily lend itself to obtaining quantitativeinvestigated because (1) NaN is known to have data. Nevertheless, CAM staining reliably confirmed3

adverse effects on skin viability and may therefore the results obtained through the MTT assay. Addi-affect buccal tissue viability, (2) buccal mucosa has tionally, contrary to SEM/TEM, it allows the directa high bacterial load, and (3) media for buccal work visualization of unfixed and viable samples, which,usually contain high levels of glucose, which pro- while not directly valuable in this particular study,motes bacterial growth. Although it appeared to have could potentially be very informative in other in-a significant effect at some time points, the addition stances [6]. For example, one can see how CAMof 0.02% sodium azide to PBS or KBR did not staining could be conducted in parallel with the use


of a second probe (an enzyme maker or a diffusion Based on the results of this study, we are currentlyprobe excitable by the 568-nm or 647-nm line of the conducting our in vitro diffusion experiments at 348CKrAr laser) to ensure tissue viability during confocal with KBR for no longer than 8 h post-mortem. Asimaging of some other event of interest. mentioned briefly earlier, the question remains

Although it is beyond the scope of this paper to whether viability is directly linked to tissue integrity,address this issue in details, it should be noted that in i.e. whether diffusion pathways are altered as tissueviable CAM-stained samples, we consistently ob- viability is gradually lost. In experiments designed toserved that calcein fluorescence was much brighter at measure permeability coefficients, this may be thethe basal level than anywhere else in the tissue. only relevant issue, and already some groups haveConsequently, our CLSM assay was based primarily focused their efforts on demonstrating linearity ofon fluorescence signal from the basal level of the transport during the time frame of the experimentepithelium, which we found to be reliably consistent [30,43]. Studies conducted in our laboratory havewith the EH-1 signal and the results from the MTT shown that care should be taken in choosing theassay. Although the localization of calcein fluores- model permeant for such investigation [36,37].cence did not prevent us from drawing conclusions Moreover, as detailed earlier, for experiments withabout the viability of our biopsy samples, it was broader goals [6], this information is clearly notsufficiently intriguing that we are currently trying to sufficient.understand it in more details. Three hypotheses arecurrently being investigated in our laboratory toexplain this phenomenon: (1) there is a preferential 4. Conclusionsdiffusion pathway for CAM from the connectivetissue towards the epithelium or along the basal CLSM used in combination with CAM and EH-1membrane, and the area of bright calcein fluores- (used as ‘live’ and ‘dead’ probes respectively) is acence actually represents CAM’s diffusion front powerful tool to assay the viability of unfixed, thicktowards the surface of the epithelium; (2) CAM is buccal mucosa samples submitted to various storageable to diffuse throughout the epithelium, but is only conditions. Although CLSM cannot realistically becleaved to calcein in the lower cell layers of the the method of choice when viability is the onlyepithelium, indicating higher esterase activity in this variable of interest, it becomes a powerful tool,part of the tissue, or finally, (3) CAM is able to however, when viability probes are used in combina-diffuse throughout the whole epithelium, it is cleaved tion with other reporting probes to monitor twoby esterases inside all viable cells, but calcein events at once. The MTT assay, on the other hand,fluorescence is quenched (pH or concentration ef- proved to be an easy, reliable, sensitive, and re-fect) in the upper part of the tissue. producible assay that can be routinely performed in

A concentration-induced quenching effect is un- most laboratories. To our knowledge, it is the firstlikely considering that staining is performed using a time that this assay is used for the determination of50 mM CAM solution and that calcein self-quench- buccal tissue viability. It is a more convenienting was reported to occur at concentrations above alternative than histology, electrophysiology, or ATP100 mM. The existence of a pH gradient within the level determination, all of which were used earliertissue would certainly be of interest to drug delivery with this tissue. Recently, we have also shown it canlaboratories like ours, however, it is currently dif- be reliably used for toxicity screening purposes [6].ficult to demonstrate due to the lack of pH probes Using these two assays, we showed that buccalexcitable by a Kr /Ar laser. We are currently focusing mucosa viability is significantly altered in PBS in theon quantifying the esterase activity in various levels first 8 h following tissue collection. In KBR, viabilityof the tissue (hypothesis 2). Hot spots of enzyme was maintained well above 75% of the original valueactivities have been observed earlier in skin [13]. for about 6 h. Work is now under way in ourMoreover, recently, Gherzi et al. reported high laboratory to determine whether the loss of viabilityexpression levels of a glucose transporter in the basal of buccal tissue in PBS is associated with alterationscells of oral mucosa [42]. in in vitro permeation behavior, and vice-versa,


confocal laser scanning microscopy and MTT assay, Corneawhether maintenance of viability in KBR is synonym16(6) (1997) 666–674.with preservation of in vivo diffusion pathways

[9] R. Supino, MTT assays, Methods Mol. Biol. 43 (1995)[36,37]. 137–149.

[10] F.B. Hershey, C.N.D. Cruickshank, L.I. Mullins, The quan-titative reduction of 2,3,5-triphenyl tetrazolium chloride byskin in vitro, J. Histochem. Cytochem. 6 (1958) 191–196.

[11] H.L. Hood, M.G. Robl, R.L. Bronaugh, Skin absorptionAcknowledgementsmethodology: Improving the lipophilicity of the receptorfluid and the accuracy of the skin viability assay, Fundam.

The authors wish to thank Harolyn Hood (FDA, Appl. Toxicol. 30(1) (1996) 168.Washington DC) for sharing her MTT protocol, Ron [12] M.V. Berridge, A.S. Tan, K.D. McCoy, R. Wang, The

biochemical and cellular basis of cell proliferation assaysBaireuther and Gabriela Fuentes from UCSF Ex-that use tetrazolium salts, Biochemica 4 (1996) 15–20.perimental Surgery for providing pig tissue on a

[13] P. Boderke, H.P. Merkle, C. Cullander, M. Ponec, H.E.regular basis, and Willy Fong and Joanne Lee (UC´Bodde, Localization of aminopeptidase activity in freshly

Berkeley) for their help with some of the MTT excised human skin: direct visualization by confocal laserassays. NIH Image is a public domain program scanning microscopy, J. Invest. Dermatol. 108(1) (1997)

83–86.developed at the U.S. National Institutes of Health[14] M.E.M.J. Meuwissen, J. Janssen, C. Cullander, H.E. Jung-and available on the Internet at http: / / rsb.in-

inger, J. Bouwstra, A cross-section device to improvefo.nih.gov/nih-image/. This study was supported byvisualization of fluorescent probe penetration into the skin by

NIH-DE11275 (CC and DI), Fondation Singer Polig- confocal laser scanning microscopy, Pharm. Res. 15(2)nac (DI), and an AACP New Investigator Grant (1998) 352–356.

[15] C.A. Squier, M. Kremer, P.W. Wertz, Continuous flow(CC).mucosal cells for measuring the in-vitro permeability ofsmall tissue samples, J. Pharm. Sci. 86(1) (1997) 82–84.

[16] R.S. Gieni, Y. Li, K.T. HayGlass, Comparison of3[ H]thymidine incorporation with MTT- and MTS-basedReferences

bioassays for human and murine IL-2 and IL-4 analysis.Tetrazolium assays provide markedly enhanced sensitivity, J.

´[1] J.C. Verhoef, H.E. Bodde, A.G. de Boer, J.A. Bouwstra, H.E. Immunol. Methods 187(1) (1995) 85–93.Junginger, F.W. Merkus, D.D. Breimer, Transport of peptide [17] T. Mosmann, Rapid colorimetric assay for cellular growthand protein drugs across biological membranes, Eur. J. Drug and survival: Application to proliferation and cytotoxicityMetab. Pharmacokinet. 15(2) (1990) 83–93. assays, J. Immunol. Methods 65 (1983) 55–63.

[2] A.J. Hoogstraate, J.C. Verhoef, A. Pijpers, L.A.M.G. Van [18] J.N. Kearney, L.A. Wheldon, G. Gowland, Cryopreservation´Leengoed, J.H.M. Verhijden, H.E. Junginger, H.E. Bodde, In of skin using a murine model: validation of a prognostic

vivo buccal delivery of the peptide drug buserelin with viability assay, Cryobiology 27(1) (1990) 24–30.glycodeoxycholate as an absorption enhancer in pigs, Pharm. [19] R.F. Brown, A.R. Groves, Succinate dehydrogenase as anRes. 13(8) (1996) 1233–1237. index of thermal injury to skin, Br. J. Exp. Pathol. 54(2)

[3] M.J. Rathbone, B.K. Drummond, I.G. Tucker, The oral (1973) 117–122.cavity as a site for systemic drug delivery, Adv. Drug Deliv. [20] R. Ferrera, A. Larese, F. Berthod, J. Guidollet, C. Rodriguez,Rev. 13 (1994) 1–22. G. Dureau, A. Dittmar, Quantitative reduction of MTT by

[4] S.W. Collier, N.M. Sheikh, A. Sakr, J.L. Lichtin, R.F. hearts biopsies in vitro is an index of viability, J. Mol. Cell.Stewart, R.L. Bronaugh, Maintenance of skin viability during Cardiol. 25(9) (1993) 1091–1099.in vitro percutaneous absorption /metabolism studies, Tox- [21] R.F.R. Brown, J.V. Harvey Kemble, Tetrazolium reductase asicol. Appl. Pharmacol. 99(3) (1989) 522–533. an index of viability of stored skin, Burns 1 (1975) 179–185.

[5] P.W. Wertz, C.A. Squier, Cellular and molecular basis of [22] M. Kietzmann, W. Loscher, D. Arens, P. Maass, D. Lubach,barrier function in oral epithelium, Crit. Rev. Ther. Drug The isolated perfused bovine udder as an in vitro model ofCarrier Syst. 8(3) (1991) 237–269. percutaneous drug absorption. Skin viability and percuta-

[6] D. Imbert, C. Cullander, Assessing penetration enhancers for neous absorption of dexamethasone, benzoyl peroxide, andbuccal drug delivery: tools for simultaneous efficacy and etofenamate, J. Pharmacol. Toxicol. Methods 30(2) (1993)toxicity screening using viable tissue samples. Proceed. Int’l. 75–84.Symp. Control. Rel. Bioact. Mater., 25 (1998), 26–27. [23] R.C. Wester, J. Christoffel, T. Hartway, N. Poblete, H.I.

[7] P.L. Moore, I.C. Maccoubrey, R.P. Haugland, A rapid pH Maibach, J. Forsell, Human cadaver skin viability for in vitroinsensitive two color fluorescence viability cytotoxicity percutaneous aborption: Storage and detrimental effects ofassay, J. Cell Biol. 111(5 part 2) (1990) 58A. heat-separation and freezing, Pharm. Res. 15(1) (1998) 82–

[8] D. Imbert, C. Cullander, Assessment of cornea viability by 84.


[24] J.H. Lass, W.J. Reinhart, D.L. Skelnik, W.E. Bruner, R.P. Craane, H.E. Junginger, Localization of the permeabilityShockley, J.Y. Park, D.L. Hom, R.L. Lindstrom, An in vitro barrier inside porcine buccal mucosa: a combined in vitroand clinical comparison of corneal storage with chondroitin study of drug permeability, electrical resistance and tissuesulfate corneal storage medium with and without dextran, morphology, Int. J. Pharm. 76(1-2) (1991) 25–35.Ophthalmology 97(1) (1990) 96–103. [35] C. Bjerre, B. Sjostrom, E. Bjork, O. Camber, Pig nasal

[25] M. Morton, Corneal storage at 4 degrees Celsius in a tissue: a characterization and in vitro permeation study,chondroitin sulphate and dextran containing medium, Aust. Pharm. Res. 13(9) (1996) S–171.N.Z. J. Ophthalmol. 20(3) (1992) 211–213. [36] D. Imbert, C. Cullander, Permeability of buccal mucosa in

[26] M. Canals-Imohr, J. Costa-Vila, D. Ruano-Gil, D. Pita- vitro: viability versus integrity, J. Dent. Res. 77(Suppl. A)Salorio, Comparative study of current corneal short-term, (1998) 290.medium-, and long-term preservation methods, Transplant [37] D. Imbert, C. Cullander, Buccal mucosa in vitro experiments:Proc. 27(4) (1995) 2418. II. Influence of viability on tissue permeability, J. Control.

[27] R.F. Potter, G. Peters, M. Carson, T. Forbes, C.G. Ellis, K.A. Rel., (1998) Manuscript in preparation, to be submitted.´Harris, G. DeRose, W.G. Jamieson, Measurement of tissue [38] A.J. Hoogstraate, H.E. Bodde, Methods for assessing the

viability using intravital microscopy and fluorescent nuclear buccal mucosa as a route of drug delivery, Adv. Drug Del.dyes, J. Surg. Res. 59(5) (1995) 521–526. Rev. 12 (1993) 99–125.

[28] C.A. Poole, N.H. Brookes, G.M. Clover, Keratocyte net- [39] V.A. Moore, I.W. Kellaway, P. Timmins, A. Dennis, De-works visualised in the living cornea using vital dyes, J. Cell velopment and chraracterization of an ex vivo buccal mucos-Sci. 106(2) (1993) 685–691. al model to assess solute permeability, Pharm. Res. 14(11)

[29] D. Harris, J.R. Robinson, Drug delivery via the mucous (1997) S–660.membranes of the oral cavity, J. Pharm. Sci 81(1) (1992) [40] C.A. Lesch, C.A. Squier, A. Cruchley, D.M. Williams, P.1–10. Speight, The permeability of human oral mucosa and skin to

[30] M.E. Dowty, K.E. Knuth, B.K. Irons, J.R. Robinson, Trans- water, J. Dent. Res. 68(9) (1989) 1345–1349.port of thyrotropin releasing hormone in rabbit buccal [41] A.J. Hoogstraate, S. Senel, C. Cullander, J. Verhoef, H.E.

´mucosa in vitro, Pharm. Res. 9(9) (1992) 1113–1122. Junginger, H.E. Bodde, Effects of bile salts on transport rates[31] M. Nair, Y.W. Chien, I. Buccal Delivery of Progestational and routes of FITC-labelled compounds across porcine

Steroids, Characterization of barrier properties and effect of buccal epithelium in vitro, J. Control. Rel. 40(3) (1996)penetrant hydrophilicity, Int. J. Pharm. 89(1) (1993) 41–49. 211–221.

[32] C.R. Bland, S.S. Davis, D.A. Rawlins, Evaluation of the [42] R. Gherzi, G. Melioli, M. De Luca, A. D’Agostino, M.viability of an in vitro porcine buccal mucosa preparation, Guastella, C.E. Traverso, F. D’Anna, A.T. Franzi, R. Cance-Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 18 dda, High expression levels of the ‘erythroid /brain’ type(1991) 499–500. glucose transporter (GLUT1) in the basal cells of human eye

[33] Y. Rojanasakul, L.Y. Wang, M. Bhat, D.D. Glover, C.J. conjunctiva and oral mucosa reconstituted in culture, Exp.Malanga, J.K. Ma, The transport barrier of epithelia: a Cell Res. 195(1) (1991) 230–236.comparative study on membrane permeability and charge [43] B. Polentarutti, A. Sjoberg, A. Peterson, E.-K. Anderberg,selectivity in the rabbit, Pharm. Res. 9(8) (1992) 1029– A.-L. Ungell, Viability of rat intestinal segments in vitro,1034. Proceed. Int. Symp. Control. Rel. Bioact. Mater. 24 (1997)

´[34] M.E. de Vries, H.E. Bodde, J.C. Verhoef, M. Ponec, W. 333–334.

Documents

Buccal mucosa in vitro experiments: I. Confocal imaging of vital staining and MTT assays for the determination of tissue viability