Uncoupled respiration stability of isolated pancreatic acini as a novel functional test for cell vitality

Abstract

Background. Assessment of cell viability is crucial in cell studies. Testing plasma membrane integrity is a traditional approach of evaluating cell viability. Mitochondrial functional capacity closely correlates with plasma membrane integrity and overall cell health. This study aimed to investigate whether any aspect of mitochondrial adaptive capacity in isolated pancreatic acini is associated with the quality of isolated pancreatic acini preparations, as determined by the dye exclusion method. Materials and Methods. Experiments were carried out on male Wistar rats weig­hing 250–300 g. A suspension of isolated pancreatic acini was obtained using collagenase. The rate of oxygen consumption of rat isolated pancreatic acini was measured with Clark oxygen electrode. Basal respiration of isolated pancreatic acini was recorded for approximately 2 min. Afterwards, the mitochondrial adaptive capacity was examined using FCCP in concentrations from 0.5 to 2 μM. Uncoupled respiratory stability was calculated as a ratio of respiration rate at high and low FCCP concentrations. Plasma membrane integrity was assessed with trypan blue staining. A total of 74 preparations of isolated pancreatic acini were used in this study. Results. In all experiments, 92–99 % of pancreatic acinar cells exhibited negative trypan blue staining, indicating intact plasma membranes. The basal and maximal uncoupled respiration rates were not affected by the fraction of trypan-negative cells. However, acini preparations with <less than 95 % plasma membrane integrity had significantly lower uncoupled respiration rates when exposed to a high concentration of FCCP (2 µM), indicating reduced stability of uncoupled respiration. Conclusions. Results of the study suggest that the stability of uncoupled respiration can serve as a novel metabolic functional test to complement the existing methods for assessing cell vitality.

Full text

243
UNCOUPLED RESPIRATION STABILITY OF ISOLATED PANCREATIC ACINI AS A NOVEL FUNCTIONAL TEST FOR CELL VITALITY
ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2023 • Том 17 / № 3 • С. 243–252
Biol. Stud. 2023; 17(3): 243–252 • DOI: https://doi.org/10.30970/sbi.1703.735
www.http://publications.lnu.edu.ua/journals/index.php/biology
© 2023 Anastasiia Zub et al. Published by the Ivan Franko National University of Lviv on behalf of Біологічні Cтудії / Studia
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UDC: 612.34:612.26:577.23
UNCOUPLED RESPIRATION STABILITY OF ISOLATED PANCREATIC ACINI
AS A NOVEL FUNCTIONAL TEST FOR CELL VITALITY
Anastasiia Zub , Bohdan V. Manko , Bohdan O. Manko ,
Volodymyr Manko , Andriy Babsky
Ivan Franko National University of Lviv, 4 Hrushevsky St., Lviv 79005, Ukraine
Zub, A., Manko, B. V., Manko, B. O., Manko, V., & Babsky, A. (2023). Uncoupled respiration
stability of isolated pancreatic acini as a novel functional test for cell vitality. Studia Biologica,
17(3), 243–252. doi:10.30970/sbi.1703.735
Background. Assessment of cell viability is crucial in cell studies. Testing plasma
membrane integrity is a traditional approach of evaluating cell viability. Mitochondrial
functional capacity closely correlates with plasma membrane integrity and overall cell
health. This study aimed to investigate whether any aspect of mitochondrial adaptive
capacity in isolated pancreatic acini is associated with the quality of said preparations,
as determined by the dye exclusion method.
Materials and Methods. Experiments were carried out on male Wistar rats weig hing
250–300 g. A suspension of isolated pancreatic acini was obtained using collagenase.
The rate of oxygen consumption of rat isolated pancreatic acini was measured with
Clark oxygen electrode. Basal respiration of isolated pancreatic acini was recorded for
approximately 2 min. Afterwards, the mitochondrial adaptive capacity was examined
using FCCP in concentrations from 0.5 to 2 μM. Uncoupled respiratory stability was
calculated as a ratio of respiration rate at high and low FCCP concentrations. Plasma
membrane integrity was assessed with trypan blue staining. A total of 74 preparations
of isolated pancreatic acini were used in this study.
Results. In all experiments, 92–99 % of pancreatic acinar cells exhibited nega-
tive trypan blue staining, indicating intact plasma membranes. The basal and maximal
uncoupled respiration rates were not a󰀨ected by the fraction of trypan-negative cells.
However, acini preparations with <less than 95 % plasma membrane integrity had sig-
nicantly lower uncoupled respiration rates when exposed to a high concentration of
FCCP (2 µM), indicating reduced stability of uncoupled respiration.

244 Anastasiia Zub, Bohdan V. Manko, Bohdan O. Manko, Volodymyr Manko, Andriy Babsky
ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2023 • Том 17 / № 3 • С. 243–252
Conclusions. Results of the study suggest that the stability of uncoupled respira-
tion can serve as a novel metabolic functional test to complement the existing methods
for assessing cell vitality.
Keywords: pancreas, acinar cells, viability, uncoupled respiration
INTRODUCTION
In vitro experiments involving isolated live cells, whether cultured immortalized cell
lines or primary cells obtained from living organisms, are extensively conducted in various
biological sstudies. Cell viability is a critical aspect for conducting accurate examinations
of biological processes. Impaired metabolism, disrupted ion transport, and compromised
physiological responses to stimuli are observed in damaged or necrotic cells. Traditional
approaches to evaluate cell viability include testing plasma membrane integrity using
techniques like trypan blue staining or assessing metabolic activity with methods such as
the MTT test.
Mitochondrial functional activity in cells is highly correlated with plasma membrane
integrity. In renal cells, cold injury causes disruption of cell morphology, a 70% lactate
dehydrogenase activity release, and nearly complete loss of both basal and uncou-
pled cell respiration (Cassim et al., 2022). In cardiac myocytes, hypoxia-reoxygenation
induces a reduction of maximal uncoupled respiration in parallel with an increase of cell
necrosis measured with a combination of calcein-AM and plasma membrane imperme-
ant ethidium homodimer-1 (Peger et al., 2015). In pancreatic acinar cells, hydrogen
peroxide causes a decrease in both basal and maximal respiration, while menadione
intensies basal respiration and reduces maximal uncoupled respiration; both com-
pounds also cause an increase in lactate dehydrogenase release (Armstrong et al.,
2018). Interestingly, antioxidant mitoQ also causes a signicant decrease in both uncou-
pled respiration and plasma membrane integrity of pancreatic acinar cells (Armstrong
et al., 2019). In many experimental settings, the mitochondrial damage precedes cell
death with or without the signs of plasma membrane rupture (Armstrong et al., 2019;
Bock & Tait, 2020; Manko et al., 2021). It is thus expedient to develop new highly sensi-
tive cell viability assays based on mitochondrial function evaluation.
Recently, our research group characterized the mitochondrial adaptive capacity of
pancreatic acinar cells using a comprehensive set of uncoupled respiration parameters
(Manko et al., 2019). The present study aimed to investigate whether any aspect of mito-
chondrial adaptive capacity in isolated pancreatic acini is associated with the qua lity of
isolated pancreatic acini preparations, as assessed with the dye exclusion method.
MATERIALS AND METHODS
Materials. Reagents used in experiments were purchased from Sigma-Aldrich (St.
Louis, Mo): sodium chloride, glucose, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic
acid (HEPES), soybean trypsin inhibitor, bovine serum albumin (BSA), sodium pyruvate,
glutamine, FCCP, collagenase type IV; Merck Chemicals (Burlington, Mass) – Calcium
chloride dihydrate. All other reagents were of the purest available grade.
Experimental animals. All manipulations with animals are accomplished in accor-
dance with the ’European Convention for the Protection of Vertebrate Animals used for
Experimental and other Scientic Purposes’ (Council of Europe No 123, Strasbourg
1985). Experimental protocols were approved by the Animal Care and Use Committee

245
UNCOUPLED RESPIRATION STABILITY OF ISOLATED PANCREATIC ACINI AS A NOVEL FUNCTIONAL TEST FOR CELL VITALITY
ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2023 • Том 17 / № 3 • С. 243–252
of Ivan Franko National University of Lviv (protocol No. 33-04-2023 of 03.04.2023).
Experiments were carried out on male Wistar rats (250–300 g). Animals were kept
under the standard conditions of a vivarium at a constant temperature with 12:12-h
light-dark cycle and on a basic diet. In vitro experiments were performed on pancreatic
acini isolated from animals, which were starved for 12 h prior to the experiment.
Isolation and viability assessment of pancreatic acinі. A suspension of isolated
pancreatic acini was obtained with collagenase (type IV, 0.2 mg/mL) as previously reported
(Manko et al., 2013) in basic incubation medium containing (mM): NaCl – 140.0, KCl –
4.7, CaCl2 – 1.3, MgCl2 – 1.0, HEPES – 10.0, glutamine – 2.0, sodium pyruvate – 2.0,
glucose – 10.0; BSA – 2.5 mg/mL; soybean trypsin inhibitor – 0.1 mg/mL and essential
MEM amino acid supplement; pH set at 7.4 with NaOH. Cell counting was performed with
a hemocytometer with trypan blue staining as described previously (Manko et al., 2021).
Oxygen consumption. The rate of oxygen consumption of isolated pancreatic acini
was measured with Clark oxygen electrode at 37 °C using YSI 5300 Biological oxygen
monitor (Yellow Springs Instruments) or SI929 6-channel Oxygen Meter (Strathkelvin).
Extracellular-like respiration medium contained (mM): NaCl – 140.0, KCl – 4.7, CaCl2 –
1.3, MgCl2 – 1.0, HEPES – 10.0, glucose – 10.0; BSA – 2.5 mg/mL and soybean trypsin
inhibitor – 0.1 mg/mL; pH set at 7.4 with NaOH. Protonophore FCCP was added step-
wise into the respiration chamber in 0.5 μM aliquots (2.0 μM total) to reach the maximal
uncoupled respiration rate as described earlier (Manko et al., 2019).
Statistical analysis. Results are presented as means ± S.E.M. Statistical analysis
was performed using Origin Pro 2018 (Northampton, Mass) software. The signicance
of di󰀨erence between the groups was determined with a one-way ANOVA followed by
a Turkey corrected post-hoc t-tests in case of a signicant di󰀨erence according to ANOVA.
RESULTS and DISCUSSION
In the course of this study, to ensure a high statistical power, a total of 74 prepara-
tions of isolated pancreatic acini were examined. To reduce the e󰀨ects of investigator
bias, a total of 59 of these preparations (experiment 1) were performed by one scientist
and other 15 were later reproduced by another scientist (experiment 2). The viability of
pancreatic acini was assessed with trypan blue staining immediately after the isolation
procedure. In all experiments, cell viability exceeded 92 %, while in experiment 2 it was
signicantly higher than in experiment 1 (Fig. 1, Fig. 2B). This may indicate the di󰀨er-
ence in cell preparation technique or in viable cell number estimation between the two
scientists. Cells with intact plasma membrane are not stained with trypan blue and thus
are di󰀩cult to count in large three-dimensional acini under the microscope. We have
also divided experiment 1 into two clusters based on cell viability with a 95 % cuto󰀨 as
a typical indicator of a high-quality cell preparation (Fig. 1C).
Basal respiration of freshly isolated pancreatic acini was recorded for approximately
2 min. Afterwards, titration with FCCP with 0.5 µM aliquots (nal concentrations 0.5, 1.0,
1.5 and 2.0 µM, ~2 min for each concentration) was performed to reach the maximal
uncoupled respiration rate. We have noted that the kinetics of respiration response to
FCCP varied greatly among preparations. In part of experiments, the maximal respi-
ration was already reached at 0.5–1 µM FCCP; further increase in the protonophore
concentration resulted in a marked inhibition of oxygen consumption rate (Fig. 1A). In
other cases, the respiration rate steadily increased up to 1.5–2 µM FCCP (Fig. 1B).
There were also all kinds of intermediate kinetic responses. In order to describe such

246 Anastasiia Zub, Bohdan V. Manko, Bohdan O. Manko, Volodymyr Manko, Andriy Babsky
ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2023 • Том 17 / № 3 • С. 243–252
0
50
100
150
200
0100 200300 400
Time (s)
0
50
100
150
200
0100 200300 400
Oxygen content (nmol)
Time (s)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
90 92 94 96 98 100
Stability of uncoupled respiration (r.u.)
Cell viability (%)
Experiment 2
Experiment 1,
cluster 1
Experiment 1,
cluster 2
r = 0.53
p =1.2·10-6
r = 0.47
p = 0.00017
A
C
B
Fig. 1. Dependence of uncoupled respiration stability of isolated pancreatic acini on cell viability: A, B – dif-
ferent examples of original respiration recordings, arrows indicate addition of 0.5 µM FCCP aliquot;
C – data from experiment 1 were split into cluster 1 – cell viability below 95 % (red points) and cluster
2 – cell viability over 95 % (green points); experiment 2 data form cluster 3 with cell viability over
95 % (black points). Black and blue lines are the results of linear regression of experiment 1 data
(black) and both experiments (blue) with corresponding Pearson’s r and p values. Each experiment is
presented by a separate point. Experiment 1 was performed by one scientist with a YSI 5300 biologi-
cal oxygen monitor, while experiment 2 was performed by another scientist with a SI929 6-channel
oxygen meter

247
UNCOUPLED RESPIRATION STABILITY OF ISOLATED PANCREATIC ACINI AS A NOVEL FUNCTIONAL TEST FOR CELL VITALITY
ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2023 • Том 17 / № 3 • С. 243–252
discrepancies in FCCP e󰀨ects, formerly we used [FCCP]max – the optimal FCCP concen-
tration when the maximal respiration rate is reached (Manko et al., 2019). However, the
discrete nature of this parameter renders statistical analysis problematic. In addition, it
is di󰀩cult to establish optimal concentration when neighboring values of respiration are
very close to each other. Thus, we calculated the uncoupled respiratory “stability” as
a ratio of respiration rate at high and low FCCP concentrations according to the equation:
Stability = (V1.5+V2.0)/(V0.5+V1.0),
where V is an uncoupled respiration rate with index as FCCP concentration in µM.
To test if the uncoupled respiratory stability depended on the plasma membrane
integrity, we have plotted data of the two parameters at Fig. 1. Above a horizontal
dashed line (Stability = 1.0) are experiments in which the respiration rate continued to
increase when high concentrations (1.5–2 µM) of FCCP were added. Conversely, when
high FCCP concentrations inhibited respiration, the stability was lower than 1. Clearly,
most of the datapoints (31 of 41) in cluster 1 of experiment 1 have stability values
under 1, which is not the case for cluster 2 (7 of 18) and experiment 2 (3 of 15) (Fig. 1).
Furthermore, there was a signicant moderate positive correlation between the stabi-
lity of uncoupled respiration and the number of trypan-negative cells with Pearson’s
r = 0.53 for the whole sample (n = 74, p = 0.0000012) and 0.47 for experiment 1 data
(n = 59, p = 0.0001728).
The statistical analysis of the di󰀨erences between three experimental clusters was
performed with ANOVA followed by a post-hoc Turkey test. As expected, the fraction of
trypan-positive dead cells was signicantly lower in experiment 1 cluster 2 compared
to cluster 1, and even lower in experiment 2 (Fig. 2B). The basal (no FCCP added)
and maximal respiration rates remained the same between all groups (Fig. 2A, C). It
was previously shown that basal and maximal respiration rates are proportional to cell
viability (Armstrong et al., 2018; Cassim et al., 2022). Apparently, small cell viability dif-
ferences do not signicantly a󰀨ect respiration parameters. However, the uncoupled res-
piration upon 2 µM FCCP was signicantly higher in experiment 2 compared to experi-
ment 1 cluster 1, indicating higher uncoupled respiration stability (Fig. 2A). There was
no statistical di󰀨erence between the respiration rates in Experiment 1 cluster 1 and
experiment 2, even though the experiments were performed with di󰀨erent instruments
and by di󰀨erent scientists (see Methods section) (Fig. 2C).
Our study revealed a notable association between slight di󰀨erences in cell viability,
as determined by the trypan blue exclusion test, and signicant e󰀨ects on uncoupled
respiration stability. Basal respiration of trypan-positive cells with either digitonin-perme-
abilized (Horbay et al., 2012; Manko et al., 2013; Pesta & Gnaiger, 2012; Rose et al.,
2019) or otherwise damaged (Cassim et al., 2022) plasma membrane is usually more
than 5-fold lower compared to intact cells due to metabolite e󰀪ux from cytoplasm. The
respiration of permeabilized pancreatic acinar cells can be restored by the addition of
exogenous oxidative substrates and ADP (Manko et al., 2013; Manko & Manko, 2013).
In our study, we investigated the respiration of pancreatic acini in extracellular-like res-
piration medium without ADP or Tricarboxylic acid cycle oxidative substrates. In addi-
tion, high Ca2+ and NaCl concentration in extracellular-like respiration medium is toxic
to mitochondria of pancreatic acinar cells (Maléth & Hegyi, 2016; Manko et al., 2013).
Because of this, we assume that a small fraction of trypan-positive necrotic cells has
a minimal contribution to oxygen consumption. Thus, uncoupled respiration stability is
the aspect of the viable trypan-negative cell population metabolism.

248 Anastasiia Zub, Bohdan V. Manko, Bohdan O. Manko, Volodymyr Manko, Andriy Babsky
ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2023 • Том 17 / № 3 • С. 243–252
Fig. 2. E󰀨ect of isolated pancreatic acini viability on their basal and uncoupled respiration: A – respiration
rates of pancreatic acini in cluster 1 (E1C1) and 2 (E1C2) of experiment 1, and experiment 2 (E2)
upon uncoupling with FCCP in increasing concentrations (x axis shows the resulting FCCP concen-
tration in the respiration chamber with 0.5 µM stepwise titration); B – the di󰀨erence in pancreatic
acinar cell necrosis in three groups; C – the maximal uncoupled respiration of pancreatic acinar
cells; * – signicant di󰀨erence vs E1C1, P ≤ 0.05, # – signicant di󰀨erence vs E1C2, P ≤ 0.05, n = 41
(E1C1), 18 (E1C2) and 15 (E2), data are shown as M ± S.E.M.
We have previously established that the uncoupled respiration stability in iso-
lated pancreatic acini depends on the type of oxidative substrate added to the medium
(Manko et al., 2019). We observed that the rapid decline in uncoupled respiration can
be attributed to the limited accumulation of certain oxidative substrates (such as suc-
cinate, α-ketoglutarate, and malate) due to the low plasma membrane permeability of
0.0
0.1
0.2
0.3
0.4
0.5
0.6
-0.5 00.5 11.5 2 2,5
Respiration rate,
nmol O2 / (106 cells·s)
[FCCP], µM
E1C1
E1C2
E2
*
0
2
4
6
8
10
Cell necrosis (%)
E1C1 E1C2 E2
*
* #
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Maximal respiratory capacity,
nmol O2 / (106 cells·s)
E1C1 E1C2 E2
A
BC

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UNCOUPLED RESPIRATION STABILITY OF ISOLATED PANCREATIC ACINI AS A NOVEL FUNCTIONAL TEST FOR CELL VITALITY
ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2023 • Том 17 / № 3 • С. 243–252
intact pancreatic acinar cells (Manko et al., 2013, 2019), Interestingly, we found that
the ester forms of these substrates (such as monomethyl-succinate and dimethyl-α-
ketoglutarate), which can penetrate the cell membrane, supported higher stability of
uncoupled respiration. Therefore, the decrease in uncoupled respiration stability in less
viable acini preparations is most likely a result of depletion of intracellular oxidative sub-
strates, which cannot be rapidly replenished by glycolysis alone.
MTT assay is a conceptually similar approach to studying cell metabolic activity, as
it relies on the activity of NADPH-dependent oxidoreductases. In human trophoblast-
like cells, the decrease in maximal uncoupled respiration upon action of linoleic acid
correlates with MTT test results (Shrestha et al., 2019). However, in astrocytes, gluta-
mate treatment caused a decrease in maximal uncoupled respiration, but did not a󰀨ect
the MTT assay (Yan et al., 2017). The direct comparison of sensitivity and reliability of
the uncoupled respiration and MTT assay is a subject for further studies. An important
issue of interpretation of results of the uncoupled respiration and other cell metabolism
tests is that the changes of metabolic parameters are often not related to cell viability.
The pitfalls of MTT assay related to the use of metabolic inhibitors in experiments have
been discussed recently (Stepanenko & Dmitrenko, 2015). Similarly, we reported that
the decrease of maximal uncoupled respiration in pancreatic acinar cells oxidizing glu-
tamine was not accompanied by any changes in cell viability (Manko et al., 2021).
CONCLUSION
The ndings of this study lead to several important conclusions. Firstly, it is advis-
able to use only preparations of pancreatic acini with high viability (> 95 % trypan-nega-
tive) in experiments to avoid the e󰀨ects of negative metabolic shift associated with lower
quality preparations. However, further investigations are required to determine whether
this conclusion holds true for other cell types and to establish appropriate viability
thresholds. Secondly, the conventional assessment of maximal uncoupled respiration
rate proves to be insu󰀩ciently sensitive in detecting mitochondrial and cellular damage.
We have established that the stability of uncoupled respiration, i.e., the ability of mito-
chondria to sustain a high respiration rate with an increasing uncoupler concentration,
is more sensitive to cell damage caused by isolation procedures. It is still unknown if
early mitochondrial and cell damage induced by toxins or other factors can be detected
similarly. Thirdly, relying solely on a single approach (plasma membrane integrity or cell
metabolic activity) is inadequate for comprehensive cell viability and functional integrity
evaluation. We propose using the stability of uncoupled respiration as a novel metabolic
functional test to complement the existing methods for assessing cell viability.
COMPLIANCE WITH ETHICAL STANDARDS
Conict of Interest: The authors have no competing interests to declare that are
relevant to the content of this article.
Human Rights: This article does not contain any studies with human subjects per-
formed by any of the authors.
Animal Rights: All experiments were carried out in accordance with the „European
Convention for the Protection of Vertebrate Animals used for Experimental and Other
Scientic Purposes” (Council of Europe No. 123, Strasbourg 1985). All animal proce-
dures were approved by the Committee for the Care and Use of Animals of the Ivan
Franko National University of Lviv under the protocol No. 33-04-2023 of 03.04.2023.

250 Anastasiia Zub, Bohdan V. Manko, Bohdan O. Manko, Volodymyr Manko, Andriy Babsky
ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2023 • Том 17 / № 3 • С. 243–252
AUTHOR CONTRIBUTIONS
Conceptualization, [M.B.O.; M.V.]; methodology, [M.B.O.]; investigation, [Z.A.;
M.B.V.]; resources, [M.V.; B.A.]; data curation, [M.B.O.]; writing – original draft prepa-
ration, [Z.A.; M.B.V., M.B.O.]; writing – review and editing, [M.V.; B.A.]; visualization,
[M.B.O.] supervision, [M.B.O., M.V.; B.A.]; project administration, [M.V.; B.A.]; funding
acquisition, [–].
All authors have read and agreed to the published version of the manuscript.
REFERENCES
Armstrong, J. A., Cash, N. J., Ouyang, Y., Morton, J. C., Chvanov, M., Latawiec, D., Awais, M.,
Tepikin, A. V., Sutton, R., & Criddle, D. N. (2018). Oxidative stress alters mitochondrial
bioenergetics and modies pancreatic cell death independently of cyclophilin D, resulting
in an apoptosis-to-necrosis shift. Journal of Biological Chemistry, 293(21), 8032–8047.
doi:10.1074/jbc.ra118.003200
Crossref ● PubMed ● PMC ● Google Scholar
Armstrong, J., Cash, N., Morton, J., Tepikin, A., Sutton, R., & Criddle, D. (2019). Mitochondrial
targeting of antioxidants alters pancreatic acinar cell bioenergetics and determines cell fate.
International Journal of Molecular Sciences, 20(7), 1700. doi:10.3390/ijms20071700
Crossref ● PubMed ● PMC ● Google Scholar
Bock, F. J., & Tait, S. W. G. (2020). Mitochondria as multifaceted regulators of cell death. Nature
Reviews Molecular Cell Biology, 21(2), 85–100. doi:10.1038/s41580-019-0173-8
Crossref ● PubMed ● Google Scholar
Cassim, S., Martin, P.-Y., & Pascolo-Rebouillat, E. (2022). ADD10 protects renal cells from
cold injuries by improving energy metabolism. Biochemical and Biophysical Research
Communications, 634, 62–69. doi:10.1016/j.bbrc.2022.10.009
Crossref ● PubMed ● Google Scholar
Horbay, R. O., Manko, B. O., Manko, V. V., Lootsik, M. D., & Stoika, R. S. (2012). Respiration
characteristics of mitochondria in parental and giant transformed cells of the murine Nemeth–
Kellner lymphoma. Cell Biology International, 36(1), 71–77. doi:10.1042/cbi20110017
Crossref ● PubMed ● Google Scholar
Maléth, J., & Hegyi, P. (2016). Ca2+ toxicity and mitochondrial damage in acute pancreatitis:
translational overview. Philosophical Transactions of the Royal Society B: Biological Sciences,
371(1700), 20150425. doi:10.1098/rstb.2015.0425
Crossref ● PubMed ● PMC ● Google Scholar
Manko, B. O., Bilonoha, O. O., & Manko, V. V. (2019). Adaptive respiratory response of rat
pancreatic acinar cells to mitochondrial membrane depolarization. The Ukrainian Biochemical
Journal, 91(3). doi:10.15407/ubj91.03.034
Crossref ● Google Scholar
Manko, B. O., Bilonoha, O. O., Voloshyn, D. M., Zub, A. M., Ivasechko, I. I., & Manko, V. V. (2021).
Pyruvate and glutamine dene the e󰀨ects of cholecystokinin and ethanol on mitochondrial
oxidation, necrosis, and morphology of rat pancreatic acini. Pancreas, 50(7). doi:10.1097/
mpa.0000000000001864
Crossref ● PubMed ● Google Scholar
Manko, B. O., Klevets, M. Yu., & Manko, V. V. (2013). An implication of novel methodology to
study pancreatic acinar mitochondria under in situ conditions. Cell Biochemistry and Function,
31(2), 115–121. doi:10.1002/cbf.2864
Crossref ● PubMed ● Google Scholar
Manko, B. O., & Manko, V. V. (2013). Inuence of Са2+ on kinetic parameters of pancreatic
acinar mitochondria in situ respiration. The Ukrainian Biochemical Journal, 85(4), 48–60.
doi:10.15407/ubj85.04.048 (In Ukrainian)
Crossref ● PubMed ● Google Scholar

251
UNCOUPLED RESPIRATION STABILITY OF ISOLATED PANCREATIC ACINI AS A NOVEL FUNCTIONAL TEST FOR CELL VITALITY
ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2023 • Том 17 / № 3 • С. 243–252
Pesta, D., & Gnaiger, E. (2012). High-resolution respirometry: OXPHOS protocols for human cells
and permeabilized bers from small biopsies of human muscle. In: C. Palmeira & A. Moreno
(Eds.), Mitochondrial bioenergetics. Methods in molecular biology (Vol 810, pp. 25–58).
Humana Press. doi:10.1007/978-1-61779-382-0_3
Crossref ● PubMed ● Google Scholar
Peger, J., He, M., & Abdellatif, M. (2015). Mitochondrial complex II is a source of the reserve
respiratory capacity that is regulated by metabolic sensors and promotes cell survival. Cell
Death & Disease, 6(7), e1835–e1835. doi:10.1038/cddis.2015.202
Crossref ● PubMed ● PMC ● Google Scholar
Rose, S., Carvalho, E., Diaz, E. C., Cotter, M., Bennuri, S. C., Azhar, G., Frye, R. E., Adams, S. H.,
& Børsheim, E. (2019). A comparative study of mitochondrial respiration in circulating blood
cells and skeletal muscle bers in women. American Journal of Physiology-Endocrinology
and Metabolism, 317(3), E503–E512. doi:10.1152/ajpendo.00084.2019
Crossref ● PubMed ● Google Scholar
Shrestha, N., Cu󰀨e, J. S. M., Holland, O. J., & Perkins, A. V. (2019). Linoleic acid increases
prostaglandin E2 release and reduces mitochondrial respiration and cell viability in
human trophoblast-like cells. Cellular Physiology and Biochemistry, 52(1), 94–108.
doi:10.33594/000000007
Crossref ● PubMed ● Google Scholar
Stepanenko, A. A., & Dmitrenko, V. V. (2015). Pitfalls of the MTT assay: direct and o󰀨-target
e󰀨ects of inhibitors can result in over/underestimation of cell viability. Gene, 574(2), 193–203.
doi:10.1016/j.gene.2015.08.009
Crossref ● PubMed ● Google Scholar
Yan, X., Shi, Z. F., Xu, L. X., Li, J. X., Wu, M., Wang, X. X., Jia, M., Dong, L. P., Yang, S. H., &
Yuan, F. (2017). Glutamate impairs mitochondria aerobic respiration capacity and enhances
glycolysis in cultured rat astrocytes. Biomedical and Environmental Sciences, 30(1), 44–51.
doi:10.3967/bes2017.005
Crossref ● PubMed ● Google Scholar
СТАБІЛЬНІСТЬ РОЗ’ЄДНАНОГО ДИХАННЯ ІЗОЛЬОВАНИХ АЦИНУСІВ
ПІДШЛУНКОВОЇ ЗАЛОЗИ ЯК НОВИЙ ФУНКЦІОНАЛЬНИЙ ТЕСТ
ЖИТТЄЗДАТНОСТІ КЛІТИН
Анастасія Зуб, Богдан В. Манько, Богдан О. Манько,
Володимир Манько, Андрій Бабський
Львівський національний університет імені Івана Франка
вул. Грушевського, 4, Львів 79005, Україна
Вступ. Аналіз життєздатності має вирішальне значення у клітинних дослі-
дженнях. Перевірка цілісності плазматичної мембрани є традиційним підходом
для визначення життєздатності клітин. Функціональна здатність мітохондрій тісно
корелює з цілісністю плазматичної мембрани та загальною інтактністю клітини.
Мета цього дослідження полягала в тому, щоби перевірити, чи пов’язані пара-
метри мітохондріальної адаптивної здатності ізольованих панкреатичних ацинусів
підшлункової залози із якістю препаратів ізольованих ацинарних клітин, визначе-
ної за допомогою фарбування трипановим синім.
Матеріали та методи. Досліди проводили на щурах-самцях лінії Wistar масою
250–300 г. Cуспензію ізольованих панкреатичних ацинусів отримували з вико-
ристанням колагенази. Швидкість споживання кисню ізольованими ацинусами

252 Anastasiia Zub, Bohdan V. Manko, Bohdan O. Manko, Volodymyr Manko, Andriy Babsky
ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2023 • Том 17 / № 3 • С. 243–252
Re ce ive d / Одержа но Revision / Доопрацьовано Accepted / Прийнято Published / Опубліковано
19 May, 2023 03 July, 2023 31 August, 2023 27 September, 2023
підшлункової залози щурів вимірювали за допомогою кисневого електрода Кларка.
Базальне дихання ізольованих ацинусів підшлункової залози реєстрували при-
близно протягом 2 хв. Після цього досліджували адаптивну здатність мітохондрій
за допомогою FCCP у концентраціях від 0,5 до 2 мкмоль/л. Незв’язану респіра-
торну стабільність розраховували як співвідношення частоти дихання за високих
і низьких концентрацій FCCP. Цілісність плазматичної мембрани оцінювали за
допомогою фарбування трипановим синім. У цьому дослідженні використано
74 різних препарати ізольованих ацинарних клітин підшлункової залози.
Результати. У всіх експериментах 92–99 % ацинарних клітин підшлункової
залози були трипан-негативними. Базальне та максимальне роз’єднане дихання не
залежали від фракції трипан-негативних клітин. Проте клітинні препарати з <95 %
цілісністю плазматичної мембрани мали значно нижчу швидкість роз’єднаного
дихання за високої (2 мкМ) концентрації FCCP, таким чином демонструючи низьку
стабільність роз’єднаного дихання.
Висновки. Ми пропонуємо стабільність роз’єднаного дихання як новий тест
метаболічної функції, що доповнює наявні методи оцінки життєздатності клітин.
Ключові слова: підшлункова залоза, ацинарні клітини, життєздатність,
роз’єднане дихання