Biochemical and Biophysical Research Communications 496 (2018) 429e435

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Quality assessment of cellular and tissue-based products using liquid
chromatography-tandem mass spectrometry
Ayako Tominaga a, Masato Sato b, *, Takumi Takahashi b, Eriko Toyoda b, Kenichi Toyoda c,Takashi Suzuki c, Masatoshi Takahashi c, Masahiko Watanabe b, Ken Okazaki aabc

Department of Orthopaedic Surgery, Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan
Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan
Shimadzu Corporation Analytical & Measuring Instruments Division, 1 Kuwabaracho, Nishinokyo, Nakagyo-ku, Kyoto, 604-8511, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 22 December 2017
Accepted 1 January 2018
Available online 4 January 2018

We are currently conducting clinical research on cell sheets for cartilage regeneration. One issue with the
future use of chondrocyte sheets as cellular and tissue-based products is quality assessment. Currently,
chondrocyte sheets are evaluated using invasive methods that cannot be performed on every sheet
produced. We report here on our liquid chromatography-tandem mass spectrometry (LC-MS/MS)
technique that allows the noninvasive assessment of every sheet using only 50 ml of culture medium. We
found that LC-MS/MS could be used to conﬁrm cell sheet viability through the measurement of glucose
and glutamine uptake, to estimate extracellular matrix production by measuring serine consumption, toestimate cell kinetics by measuring cytidine and uracil concentrations, and to estimate melanoma
inhibitory activity level by measuring pyridoxal concentration. LC-MS/MS may be useful for the noninvasive assessment of products to be used in regenerative medicine.
© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

Keywords:
Chondrocyte sheet
Noninvasive assessment technique
Liquid chromatography-tandem mass
spectrometry (LC-MS/MS)
Regenerative medicine

1. Introduction
We are currently conducting clinical research on cell sheets for
cartilage regeneration [1]. One future issue with the use of chondrocyte sheets [2] as cellular and tissue-based products is quality
assessment. Currently, both rabbit and human chondrocyte sheets
are evaluated by the same methods, which are all invasive methods
that cannot be performed on every sheet produced. However, it is
now possible to use liquid chromatography-tandem mass spectrometry (LC-MS/MS), a technology recently developed by Shimadzu Corporation, to assess chondrocyte sheet quality. We havefound that LC-MS/MS can be used to perform a noninvasive
assessment of every sheet. This paper describes the use of LC-MS/

Abbreviations: LC-MS/MS, Liquid Chromatography-tandem mass spectrometry;
DMEM, Dulbecco's modiﬁed Eagle's medium; FBS, fetal bovine serum; ECM,
extracellular matrix; ELISA, enzyme-linked immunosorbent assay; HE, hematoxylin
and eosin; Col, collagen; AGCN, aggrecan; MIA, melanoma inhibitory activity; TGFb1, transforming growth factor-b1.
* Corresponding author. Department of Orthopaedic Surgery, Surgical Science,
Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 2591193, Japan.
E-mail address: sato-m@is.icc.u-tokai.ac.jp (M. Sato).

MS to analyze chondrocyte sheet production and to determine
the optimal number of cell layers for rabbit chondrocyte sheets. We
also describe our experiments to determine whether humanchondrocyte sheets show similar results.
2. Materials and methods
All animal experiments were conducted in accordance with
“The Guide for the Care and Use of Laboratory Animals” publishedby the National Institutes of Health as well as Tokai University's
animal handling code of conduct. Experiments using humanchondrocytes were conducted after receiving approval from Tokai
University's medical ethics committee. LC-MS/MS analysis was
conducted after ﬁrst using modern methods to assess the histological and biochemical characteristics of previously prepared
rabbit chondrocyte sheets laminated with one, three, or six cell
layers.
2.1. Preparation of cartilage sheets
2.1.1. Cell collection/cell sheet preparation
Chondrocytes and synovial cells were collected from both kneesof 10 female Japanese white rabbits (16e18 weeks old, 3 kg average

https://doi.org/10.1016/j.bbrc.2018.01.001
0006-291X/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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body weight; Tokyo Laboratory Animals Science Co., Tokyo, Japan)
for use in rabbit chondrocyte sheets. Chondrocytes and synovial
cells for human cartilage sheets were collected from patients aged
20e80 years who underwent surgery at Tokai University; three ofthe patients underwent total knee arthroplasty surgery, and one
patient underwent surgery for a traumatic knee injury.
Collagenase type 1 (Worthington, Lakewood, NJ, USA) was dissolved in Dulbecco's modiﬁed Eagle's medium/F12 (DMEM/F12;
Gibco, Grand Island, NY, USA) and agitated for 4 h (37  C, 5% CO2,
20% O2). The culture medium was ﬁltered after agitation using a
100-mm cell strainer (BD Falcon Labware, Franklin Lakes, NJ, USA).
Synovial cells were cultured in DMEM/F12 with 10% fetal bovine
serum (FBS; Gibco) and 1% antibioticeantimycotic mixture (Gibco),and passaged until P1. Additionally, on culture day 4, ascorbic acid
(Wako Junyaku Kogyo, Osaka, Japan) was added to a ﬁnal concentration of 0.1% in the culture medium (37  C, 5% CO2). Human synovial cells were cultured by following the same protocol, except
with 20% FBS (Ausgenex, Oxenford, Australia).
After isolating chondrocytes using collagenase, the cells were
cryopreserved. Synovial cells were thawed directly before use.
When both cell types were ready, they were cocultured. Chondrocytes were cultured on temperature-responsive cell culture
inserts (4.2 cm2; CellSeed Inc., Tokyo, Japan), and synovial cells
were seeded on a companion plate (9.6 cm2; BD Falcon).
Temperature-responsive cell culture inserts (UpCell; CellSeed)
were made using poly(N-isopropylacrylamide), which undergoes atemperature-dependent and reversible transition from being hydrophobic to hydrophilic, which changes the surface properties of
the insert in response to a change in temperature [3] Use of thesecell culture inserts allows the removal of cells and the extracellularmatrix (ECM) in sheet form by simply adjusting the temperature
without the need for enzymes. Cells were cocultured for 14 days ata seeding density of 50000 cells/cm2 for chondrocytes and10000 cells/cm2 for synovial cells [4]. We have previously shownthat coculturing chondrocytes and synovial cells increases chondrocyte proliferation [5e7].
2.1.2. Preparation of laminated sheets
After 14 days of coculture, the temperature-responsive cell
culture plates were placed at 25  C for 30 min, and the sheets werethen collected. The inserts were removed, and the chondrocyte
sheets were manually laminated using polyvinylidene ﬂuoride
rings. Rabbit chondrocyte sheets were made by laminating with
one, three, or six layers, and human chondrocyte sheets were madeby laminating with three layers. After lamination, rabbit chondrocyte sheets were cultured in a cell culture dish (4 ¼ 10 cm) in
DMEM/F12 with 10% FBS, 1% antibioticeantimycotic mixture, and
0.1% ascorbic acid. Human chondrocyte sheets were cultured in thesame way using DMEM/F12 with 20% FBS, 1% antibioticeantimycotic mixture, and 0.1% ascorbic acid. The culture
medium was replaced every 72 h, and the cells were cultured for 7
days.
2.2. Assessment of chondrocyte sheets
Rabbit chondrocyte sheets were assessed by immunostaining.
The component cells were counted, and the concentrations of humoral factors were assessed by enzyme-linked immunosorbent
assay (ELISA) and LC-MS/MS. For human chondrocyte sheets, thecomponent cells were counted, and the concentrations of humoral
factors were assessed by ELISA and LC-MS/MS.

Finetechnical Co. Ltd. Tokyo, Japan), and sliced into 0.8-mm-thickcryosections at 80  C. The tissue sections were stained with 0.1%
safranin O and hematoxylin and eosin (HE) dye (n ¼ 3). Immunostaining was then performed using antibodies to type I collagen(Col I), type II collagen (Col II), and aggrecan (AGCN). Goat IgG(200 mg/ml), goat anti-human Col I (400 mg/ml), and goat antihuman AGCN (Santa Cruz Biotechnology, Inc., Santa Cruz, CA,
USA) were used for Col I and AGCN, respectively. Mouse IgG1(100 mg/ml) and anti-Col II mIgG1 (Daiichi Fine Chemical Co. Ltd.,
Toyama, Japan) were used for Col II (n ¼ 3). The sections were thenwashed with phosphate-buffered saline and stained with biotinylated polyclonal anti-mouse immunoglobulin (1/600; Dako,
Glostrup, Denmark). The other sections were incubated with PBS
instead of speciﬁc primary antibodies and stained as a negative
control.
2.2.2. Cell counting
Whole laminated rabbit chondrocyte sheets were dissolved in
10% FBS and collagenase type I (37  C, 1 h). The cells were counted
after counterstaining with trypan blue (Thermo Fisher Scientiﬁc,Waltham, MA, USA). Cells were counted on days 0, 3, and 7 afterlamination. Measurements on day 0 were conducted by measuring
single-layer sheets, and multiplying the results by 3 or 6 instead of
measuring the other groups (n ¼ 3). Human chondrocyte sheets
were dissolved on day 7 after lamination using TrypLE Express(Gibco, 37  C, 15 min) and collagenase P (Roche, Basel, Switzerland)for 30 min at 37  C, and then counterstained following the same
protocol as that for rabbit chondrocyte sheets for cell counting(n ¼ 4).
2.2.3. ELISA
After 7 days of culture after lamination, DMEM/F12 was prepared with 1% FBS and 1% antibioticeantimycotic, and 3 ml of thismedium was added to the cell culture plates. The sheets weretransferred to the plates and cultured for 72 h, and the concentrations of melanoma inhibitory activity (MIA) and transforming
growth factor-b1 (TGF-b1) secreted into the culture media were
measured using ELISA. MIA concentration was measured using a
96-well plate ELISA kit (Roche, Mannheim, Germany), and TGF-b1
concentration was measured using a Quantikine Human TGF-b1
ELISA Kit (R&D Systems, Minneapolis, MN, USA) (rabbit: n ¼ 3 each;human: n ¼ 4 each).
2.2.4. LC-MS/MS analysis
LC-MS/MS was performed using only 50 ml of the cell culture
supernatant. A High-performance liquid chromatography systems(Nexera X2, LCMS-8050 and LC/MS/MS Method Package for Cell
Culture Proﬁling; Shimadzu Corp, Kyoto, Japan) were used, and thedata were analyzed using the LabSolutions LCMS. The results areshown as the area ratio between the 2-isopropylmalic acid reference standard and the target molecules. During the culture of thelaminated sheets, the culture medium was replaced after 72 h (onday 3) and 144 h (on day 6), when the replaced culture medium wascollected for LC-MS/MS analysis from each sample (rabbit: n ¼ 3;
human n ¼ 4). Additionally, DMEM/F12 with 10% FBS, 1% antibioticeantimycotic mixture, and 0.1% ascorbic acid was measured
as a blank sample. Similarly, for human chondrocyte sheets, DMEM/
F12 with 20% FBS, 1% antibioticeantimycotic mixture, and 0.1%
ascorbic acid was measured as a blank. For clarity, the data areshown after subtracting the value for the relevant blank sample.
2.3. Statistical analysis

2.2.1. Immunohistological assessment
Rabbit chondrocyte sheets were cultured for 7 days after lamination, embedded using OTC compound 4583 (Sakura

ELISA results were analyzed using a four-parameter logistic
curve. All other analyses were performed using EZR statistical

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software (Easy R; Saitama, Japan) [8]. One-way ANOVA was used tocompare the rabbit chondrocyte sheets between groups. Pearson's
product-moment correlation coefﬁcient was used to analyze therelationships between different components detected by LC-MS/
MS.

431

samples. Tissue thickness was calculated as the sum of the sheet
thicknesses. Three-layer sheets were the thickest. The layers in the
six-layer sheets did not adhere to each other and were muchthinner than the cell layer in the single-layer sheet. The thicknesses
were as follows: single-layer sheet ¼ 51.9 ± 7.0 mm, three-layer
sheet ¼ 116.6 ± 6.5 mm, six-layer sheet ¼ 81.7 ± 9.4 mm.

3. Results
As a preface to the experiments using rabbit chondrocytes, wenote that they naturally proliferate and repeatedly form laminatedlayers on their own, whereas human chondrocytes do not exhibit
this behavior.
3.1. Tissue thickness
Rabbit chondrocyte sheets were assessed in HE-stained

3.2. Immunohistological analysis
Rabbit chondrocyte sheets were stained with safranin O for
assessment. Single-layer sheets stained most strongly (Fig. 1A), butthree-layer and six-layer sheets also stained positively (Fig. 1B and
C). All sheets stained negatively for Col I (Fig. 1DeG) but positively
for Col II (Fig. 1HeK) and AGCN (Fig. 1LeO). There was no clear
difference in staining pattern between the sheets with different
numbers of layers.

Fig. 1. Histological and biochemical results of the invasive examination. (AeC) Results of safranin O staining. A, B, and C correspond to single-, three-, and six-layer chondrocyte
sheets, respectively. Single-layer sheets stained strongly for safranin O; three- and six-layer sheets stained positively but not as strongly. The line represents 50 mm. (DeG) Results ofcollagen I (Col I) immunostaining. D, E, F, and G correspond to control, single-, three-, and six-layer chondrocyte sheets, respectively. All sheets stained negatively for Col I. (HeK)
Results of Col II immunostaining. H, I, J, and K correspond to control, single-, three-, and six-layer chondrocyte sheets, respectively. All sheets stained positively for Col II. (LeO)
Results of aggrecan (AGCN) immunostaining. L, M, N, and O correspond to control, single-, three-, and six-layer chondrocyte sheets, respectively. All sheets stained positively for
AGCN. (P) Cell number (  105 cells). Day 0: single-layer ¼ 6.9 ± 1.3, three-layer ¼ 20.7 ± 1.3, and six-layer ¼ 41.4 ± 1.3. Day 3: single-layer ¼ 3.6 ± 0.5, three-layer ¼ 12.1 ± 0.8, and sixlayer ¼ 7.7 ± 1.2. Day 7: single-layer ¼ 12.3 ± 1.6, three-layer ¼ 35.1 ± 5.5, and six-layer ¼ 22.7 ± 2.2. The vertical axis represents cell count (  105), and the horizontal axis represents days. (Q) Whole-sheet MIA production (ng/ml): single-layer ¼ 101.92 ± 3.19, three-layer ¼ 97.36 ± 11.63, and six-layer ¼ 63.32 ± 5.19. (R) TGF-b1 production (ng/ml): singlelayer ¼ 2.39 ± 0.21, three-layer ¼ 2.03 ± 0.16, and six-layer ¼ 1.99 ± 0.22. The vertical axis represents concentration (ng/ml). MIA concentration differed signiﬁcantly between singlelayer and six-layer chondrocyte sheets and between three-layer and six-layer chondrocyte sheets.

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A. Tominaga et al. / Biochemical and Biophysical Research Communications 496 (2018) 429e435

Fig. 2. Results of the cell culture proﬁling system. (A) Glucose, serine, and glutamine used in all groups. The vertical axis represents the area ratio, which was calculated as thetarget substance area value divided by the standard substance area value. The horizontal axis represents days. (B) The concentrations of these substances differed by >30% between
the three groups. In the six-layer sheets, these concentrations were high on day 3 and low on day 6. Thymine and deoxycytidine were exceptions, and their concentrations were low
on day 3 and high on day 6. The vertical axis represents the area ratio, calculated as described above. The horizontal axis represents days. (C) Comparison between rabbit and humanchondrocyte sheets. All results are shown for three-layer rabbit and human chondrocyte sheets on day 7. The vertical axis represents units. (D) Comparison of three-layer rabbit andhuman sheets. The peaks for glucose, serine, and glutamine concentrations occurred on different days in the rabbit and human. The vertical axis represents the area ratio, which was
calculated as described in Fig. 1. The horizontal axis represents days.

3.3. Cell counts
The cell count initially decreased and then increased in every
group. The three-layer sheets had the highest ﬁnal cell count,whereas the six-layer sheet exhibited the largest change over time
(Fig. 1P). The cell count for human chondrocyte sheets was
1.60 ± 0.22 (  105 cells). The cell counts were much higher insheets made from rabbit chondrocytes than in those made fromhuman chondrocytes (Fig. 2C).
3.4. Humoral factor production by chondrocyte sheets
For rabbit chondrocyte sheets, MIA secretion differed between
single-layer and six-layer sheets and between three-layer and sixlayer sheets (P < .05 for both comparisons; Fig. 1Q). However, TGFb1 secretion did not differ signiﬁcantly between the number oflayers (Fig. 1R). The concentrations produced by human chondrocyte sheets were: MIA ¼ 16.72 ± 5.54 ng/ml and TGFb1 ¼1.27 ± 0.31 ng/ml. MIA and TGF-b1 concentrations were higher
in rabbit chondrocyte sheets than in human chondrocyte sheets(Fig. 2C).
3.5. Mass spectrometry results
LC-MS/MS detected 95 distinct molecules (Table 1). Of these, 58
molecules differed between the samples and the blank sample. The
molecules that exhibited large changes were examined and aremarked with an asterisk in Table 1. Glucose, pyruvic acid, isocitricacid, succinic acid, serine, and glutamine were consumed in large

quantities by the chondrocyte sheets. Lactic acid, citric acid, fumaricacid, and malic acid were produced in large quantities by thechondrocyte sheets. Most of these substances are closely related toenergy production by glycolysis and the citric acid cycle.
There were large differences in the concentrations of the nucleic
acid-related molecules cytidine, uridine, inosine, guanosine,
xanthine, hypoxanthine, uracil, thymine, deoxycytidine, and putrescine (an amine) between the experimental groups (Fig. 2B). The
molecules that were highly active in human chondrocyte sheets
were roughly the same as those in rabbit chondrocyte sheets.
However, putrescine activity was higher in rabbit than in humanchondrocyte sheets (P < .05) (Fig. 2D).
4. Discussion
Multiple mechanisms contribute to cartilage tissue repair/
regeneration by cell sheets, including tissue repair by cell sheetderived free chondrocytes, supply of growth factors from the cellsheet, and the role of the cell sheet as a barrier that inhibits thepenetration of catabolic factors [9]. Histological analysis showedthat every sheet stained successfully with safranin O, and immunostaining showed that all sheets stained positively for Col II and
AGCN. However, the three-layer sheets were the thickest and had
the highest cell count. All sheets exhibited a transient decrease incell count on day 3 and an increase by day 7. This transient decreasein cell count may reﬂect the change in the cell culture environment
from the temperature-responsive cell culture insert to a standard
culture dish or handling during the laminating process. Nevertheless, rabbit chondrocytes naturally proliferate and repeatedly form

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433

Table 1The 95 substances identiﬁed by the LS-MS/MS method. Substances identiﬁed in the cell culture proﬁling system. The substances marked with (*) had
signiﬁcant changes with blank.
Internal Standard
2-Isopropylmalic
SugarsGluconic acid
Glucosamine
Glucose*SucroseThreonic acid
Nucleic acid associated compounds
AdenineAdenosine
Adenosine monophosphate
Cytidine*
Cytidine monophosphate
Deoxycytidine*
GuanineGuanosine
Guanosine monophosphate
Hypoxanthine
Inosine*Thymidine
Thymine*Uracil*Uric acid
Uridine*Xanthine*
Xanthosine
Antibiotics
Penicilin G

Amino Acid and Derivatives
2-Aminoadipic acid
4-Aminobutyric acid
4-Hydroxyproline
5-Glutamylcysteine
5-Oxoproline
AlanineAlanyl-glutamine
Arginine
Asparagine
Aspartic acid
Citrulline
Cystathionine
Cysteine
CystineGlutamic acid*
Glutamine*
Glutathione
GlycineGlycyl-glutamine
Histidine
Isoleucine
Kynurenine
LeucineLysineMethionine
Methionine sulfoxide
N-Acetylaspartic acid
N-Acetylcysteine
Ornithine
Oxidized glutathione
Phenylalanine
Pipecolic acid
ProlineSerine*Threonine
Tryptophan
Tyrosine
Valinemultiple layers on their own, which may have contributed to theincrease in cell count after the initial decrease.
Our results suggest the biochemical importance of MIA and TGFb1 in chondrocyte sheets. MIA is a soluble protein that is secreted
by malignant melanoma cells and chondrocytes [10]. Humoral MIA
level is related to cartilage remodeling and can be used as an
objective marker of cartilage characteristics [10]. Stimulatingchondrocytes with TGF-b1 increases ECM production, which isrelated to the maintenance of chondrocyte homeostasis and cell
division [11]. The ELISA showed that the concentration of MIA
secreted by the sheets was signiﬁcantly higher for the single-layer
and three-layer sheets than for the six-layer sheets. These results
indicate that, from a histological and biochemical perspective, threelayers is the ideal number of layers for the chondrocyte sheets. The
results from the six-layer sheets suggest that increasing numbers oflayers limits the diffusion of nutrients through the multiple layers.
LC-MS/MS was developed as a technology to assess undifferentiated cells over time while they are being cultured. Using LC-MS/
MS, we detected 95 compounds, including sugars, nucleic acids,
proteins, and vitamins. In the discussion below, we focus on two
major categories of moleculesdthose that were consumed or
produced at a high level and those whose concentrations differed
between experimental groups.
Glucose, glutamine, and serine were all consumed in large
quantities (Fig. 2A). Glucose provides a source of energy for all
cellular activities, and glutamine is used as a nitrogen source in thebiosynthesis of various compounds [ 12,13]. These two molecules

Vitamins
4-Aminobenzoic acid
Ascorbic acid
Ascorbic acid 2-phosphate
BiotinCholineCyanocobalamin
Ergocalciferol
Folic acid
Folinic acid
Lipoic acid
Niacinamide
Nicotinic acid
Pantothenic acid
Pyridoxal*
Pyridoxine
Riboavin
Tocopherol acetate
Others2-Aminoethanol
2-Ketoisovaleric acid
3-Methyl-2-oxovaleric acid
4-Hydroxyphenyllactic acid
Citric acid*
Ethylenediamine
Fumaric acid*
Glyceric acidHistamine
Isocitric acid*
Lactic acid*
Malic acid*
O-Phosphoethanolamine
Putrescine*
Pyruvic acid*
Succinic acid*

were consumed most heavily between days 3 and 6, when the cellcount was increasing. The concentrations of these molecules areaffected by cellular activity such as cell division. Although serine is
not consumed in large quantities in ordinary cells, it exhibited
similar dynamics to glucose and glutamine, which is a characteristic response of chondrocytes to culture. Chondrocytes produce
large quantities of the ECM, which is made mainly of proteoglycans,of which serine is a major component [14]. This is a likely reason for
the increased serine consumption during the period of high cellular
activity from days 3e6.
The concentrations of molecules involved in glycolysis and thecitric acid cycle also exhibited marked increases and decreases. The
increases and decreases in glycolysis- and citric acid cycle-related
molecules in the cell culture proﬁling system are shown in Fig. 3.
The concentrations of glucose and pyruvic acid decreased; both ofthese are involved in glycolysis, and these changes reﬂect increased
glycolysis in the cells. The increased lactic acid and citric acidconcentrations may also reﬂect the increase in glycolysis. Lactic
acid concentration correlated signiﬁcantly with glucose and citricacid consumption (P < .05), providing evidence that the increased
amount of lactic acid was produced by glycolysis. However, isocitricacid and succinic acid concentrations decreased whereas fumaricacid and malic acid concentrations increased. This is not the expected citric acid cycle response, and it is possible that the activityof the enzyme that catalyzes the conversion from citric acid toisocitric acid was affected by the culture system. This enzyme is aniron sulfur protein called aconitase, which requires sufﬁcient iron to434

A. Tominaga et al. / Biochemical and Biophysical Research Communications 496 (2018) 429e435

Fig. 3. Glycolysis pathway and citric acid cycle. The arrows correspond to the content
for each substance compared with the blank. The data contained in this ﬁgure suggestthat the citric acid cycle did not operate normally in the chondrocyte sheets.

function properly [12]. Although the FBS used in our researchcontains iron, it may be useful to monitor iron levels. The low malic
acid concentration probably reﬂected abnormal conversion from
citric acid to isocitric acid, and the marked increase in fumaric acidconcentration may reﬂect its presence in the urea cycle [12,13].
Fumaric acid is produced by both the citric acid cycle and theprocessing of excess NHþ
4 from the breakdown of amino acids. The
concentration of fumaric acid measured in our system reﬂects boththe amounts produced by the citric acid cycle and synthesized fromexcess NHþ
4 from the breakdown of amino acids. This idea is supported by the observation of high fumaric acid and malic acidconcentrations on day 3, when there was signiﬁcant cell death,followed by decreased concentrations by day 6. Our ﬁndings suggest that the citric acid cycle was not functioning properly in thisculture system.
The articular cartilage is usually a hypoxic environment. Chondrocytes exhibit increased cellular activity in hypoxic conditions
with oxygen concentrations of around 2% [15]. These experiments
were conducted under 20% oxygen, so the experimental conditions
were not necessarily consistent with in vivo conditions. Additionally, glycolysis and the citric acid cycle have functions beyond energy production, and other related factors (supplements of aminoacids, nucleotides, and so on) should also be considered [16].
We found some molecules, many of which are related to nucleic
acids, whose concentrations differed markedly between theexperimental groups. The concentrations of these molecules werehigh on day 3, when there was signiﬁcant cell death, but decreased
by day 6. The concentrations were very high in the six-layer sheets,
which had the higher cell death rate, and this ﬁnding suggests thatthese molecules are related to cell death. To compare the changes inconcentrations of these molecules with the changes in cell counts,
we estimated cell counts for day 6 using the cell count data presented above. Signiﬁcant correlations between cell count and thechanges in the concentrations of cytidine, uridine, guanosine,Fig. 4. Correlation between cell count and MIA concentration. (A) Correlations between cell number and concentrations of other nucleic acid substances and putrescine in the
rabbit; all correlations were signiﬁcant (P < .05). The vertical axis represents concentration (ng/ml), and the horizontal axis represents cell number (  105). (B) In the humanchondrocyte sheets, cytidine and uracil concentrations correlated with cell number (P < .05). The vertical axis represents concentration (ng/ml), and the horizontal axis represents
cell number (  105). (C) Correlation between MIA and pyridoxal concentrations in both rabbit and human. Log (MIA) and log (pyridoxal) correlated in both the rabbit and human(P < .05).

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xanthine, hypoxanthine, and uracil were found (P < .05) (Fig. 4A).
Similar changes were seen in the concentration of putrescine (anamine), and these changes correlated with cell count (P < .05). The
details of putrescine function are not fully known, but it is involvedin amino acid breakdown in living cells and is released from deadcells [17]. In future, these molecules may be useful indicators oflarge changes in cell count.
It would be ideal to identify molecules related to cytokines, such
as MIA and TGF-b1, and to use this to assess the quality of chondrocyte sheets. We were unable to identify any molecules related to
TGF-b1, but we did observe that MIA concentration correlated withglucose and pyridoxal concentrations (P < .05). At the same time,
MIA and pyridoxal concentrations showed a signiﬁcant logarithmiccorrelation (P < .05). Pyridoxal is one form of vitamin B6 that acts as
a prosthetic group for all aminotransferases [12]. To our knowledge,
no other studies have reported a relationship between MIA andpyridoxal concentrations in cell culture.
We observed increased glucose, glutamine, and serine consumption in human chondrocyte sheets similar to that observed for
rabbit chondrocyte sheets. Cytidine and uracil concentrations
correlated with the cell count (Fig. 4 B) (P < .05). In addition, alogarithmic relationship between MIA and pyridoxal concentrations was observed (Fig. 4C) (P < .05). These results show that LCMS/MS can be used in the following ways. (1) Glucose and glutamine concentrations can be measured to screen for the biologicalactivity of sheets. (2) Serine concentration can be measured toscreen for the presence of the ECM. (3) Cytidine and uracil concentrations can be used to estimate cell kinetics. (4) Pyridoxal
concentration can be used to estimate MIA concentration. Further
research using human cells is expected to help improve thedevelopment of noninvasive methods for assessment of productquality control in regenerative medicine.
Funding
This research was supported by the Japan Agency for Medical
Research and Development through the Project Focused on
Developing Key Evaluation Technology: Manufacturing Technology
for Industrialization in the Field of Regenerative Medicine [grant
number 14525207 to M.S.].
Acknowledgements
We thank CellSeed Inc. for supplying the temperature-435

responsive culture inserts.
Transparency document
Transparency document related to this article can be foundonline at https://doi.org/10.1016/j.bbrc.2018.01.001.
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