Regenerative Therapy 14 (2020) 215e221

Contents lists available at ScienceDirect

Regenerative Therapy
journal homepage: http://www.elsevier.com/locate/reth

Original Article

Development of an efﬁcient vitriﬁcation method for chondrocyte
sheets for clinical application
Asuka Hayashi a, Miki Maehara b, Ayuko Uchikura a, Hitomi Matsunari a, c,Kazuaki Matsumura d, Suong-Hyu Hyon e, Masato Sato b, c, Hiroshi Nagashima a, c, *a

Laboratory of Medical Bioengineering, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama, Kawasaki 214-8571, Japan
Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan
Meiji University International Institute for Bio-Resource Research (MUIIBR), 1-1-1 Higashimita, Tama, Kawasaki 214-8571, JapandSchool of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 Japane
The Joint Graduate School of Veterinary Medicine, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-8580, Japanbca r t i c l e i n f o

a b s t r a c t

Article history:
Received 16 February 2020
Received in revised form
24 March 2020
Accepted 14 April 2020

Introduction: Regenerative therapy using chondrocyte sheets is effective for osteoarthritis. The clinicalapplication of chondrocyte sheet therapy is expected to be further advanced by the use of a feasible
cryopreservation technique. Previously, we developed a chondrocyte sheet vitriﬁcation method; however, it was too complex to be used for routine clinical application. Here, we aimed to develop a prototype method for vitrifying chondrocyte sheets for clinical practice.
Methods: We developed a “circulating vitriﬁcation bag” as a container to process cell sheets for vitriﬁcation in an efﬁcient and sanitary fashion. Moreover, we invented the “vitriﬁcation storage box”, which isuseful for the vitriﬁcation of cell sheets, long-term preservation, and transportation. These devices wereused to vitrify rabbit chondrocyte sheets, which were then assessed for their structural characteristics
and the viability of the component cells after rewarming.
Results: In all cell sheet samples (n ¼ 7) vitriﬁed by the circulating vitriﬁcation bag method, the integrityof the sheet structure was maintained, and the cell survival rate was similar to that of non-vitriﬁed
samples (91.0 ± 2.9% vs. 90.0 ± 3.0%). Proteoglycan and type II collagen, which are major components
of cartilage, were densely and evenly distributed throughout the chondrocyte sheet subjected to vitriﬁcation similarly to that observed in the non-vitriﬁed sheet. After long-term storage using the vitriﬁcation storage box, the cell sheets maintained normal structure and cell viability (survival rate:
81.2 ± 1.0% vs. 84.3 ± 1.8%) compared to the non-vitriﬁed sheet.
Conclusion: Our results indicate that the circulating vitriﬁcation bag method is an effective approach forrealizing the clinical application of vitriﬁed chondrocyte sheets. The vitriﬁcation storage box is also
useful for the long-term preservation of vitriﬁed cell sheets, further enhancing the feasibility of theclinical application of cryopreserved chondrocyte sheets.
© 2020, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
Chondrocyte
Cell sheet
Vitriﬁcation
Cryopreservation
Osteoarthritis

1. Introduction

Abbreviations: DMSO, dimethyl sulfoxide; EG, ethylene glycol; FBS, fetal bovine
serum; LN, liquid nitrogen; PBS, phosphate buffered saline.
* Corresponding author. Laboratory of Medical Bioengineering, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama, Kawasaki 214-8571, Japan.
E-mail address: hnagas@meiji.ac.jp (H. Nagashima).
Peer review under responsibility of the Japanese Society for Regenerative
Medicine.

Regenerative medicine using autologous chondrocyte sheets
has been reported to be effective for the treatment of osteoarthritis[1]. Allogeneic cell sheets created from polydactyly derived chondrocytes obtained from polydactyly surgery were reported to have
similar treatment effects as autologous chondrocyte sheets [2,3].
Cryopreservation of cell sheets would help expand the clinicalapplication of cell sheet therapy and promote its industrialization.
We previously developed a vitriﬁcation method that allows for the

https://doi.org/10.1016/j.reth.2020.04.006
2352-3204/© 2020, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

216

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221

cryopreservation of chondrocyte sheets [4]. Through this method,cryopreserved chondrocyte sheets were found to maintain theircartilage repair ability [5]. However, this previous method involves
complex work processes [4], and it would be desirable to improve it
to develop a simpler and more practical method for clinical application. In addition, for the expansion of the use of chondrocyte
sheet therapy, it is important to establish technologies for the longterm preservation and transportation of cryopreserved cell sheets.
To meet these requirements, we developed the “circulating vitriﬁcation bag” as a prototype device for allowing the practical use of
the chondrocyte sheet vitriﬁcation method. Moreover, as a device
that can maintain the stable vitriﬁed state of cell sheets, the“vitriﬁcation storage box” was developed. The results of the performance of these devices based on the vitriﬁcation of rabbit
chondrocyte sheets are reported.
2. Materials and methods
2.1. Preparation of rabbit chondrocyte sheets
Rabbit chondrocyte sheets were prepared according to ourprevious report [4]. Brieﬂy, commercially available primarycultured cells derived from the knee cartilage of a Japanese whiterabbit (CHC04C; Cosmo Bio Co., Ltd., Hokkaido, Japan) were plated
onto temperature-responsive culture dishes (UpCell®, diameter:
35 mm; CellSeed, Tokyo, Japan) at a density of 5.9  104 to
7.5  104 cells/dish and cultured in DMEM/F12 medium (11320;
Thermo Fisher Scientiﬁc K.K., Tokyo, Japan) supplemented with 20%
fetal bovine serum (FBS; SH30070.03, GE Healthcare, Tokyo, Japan)
at 37.5  C in a humidiﬁed atmosphere of 5% CO2 in air. Uponreaching conﬂuence, the medium in each dish was replaced with
RPMI1640 medium (11875; Thermo Fisher Scientiﬁc K.K.) supplemented with 10% FBS and 100 mM L-ascorbic acid (3140401A1039;
FUJIFILM Pharma Co., Ltd., Tokyo, Japan). The cells formed a single
thin layer 2 weeks after plating, at which time the UpCell® dishes
were placed at 25  C for 30 min to promote detachment of the cellsheet from the bottom surface of the dish. The cell sheet was thenremoved from the dish using a cell shifter (CSD001; CellSeed). Twocell sheets were layered together to form a double-layered sheet,
which was further cultured for 1 week.
2.2. Vitriﬁcation and rewarming solutions
Vitriﬁcation and rewarming solutions were prepared according
to our previous report [4] (Table 1). HEPES (20 mM)-buffered Tissue
Culture Medium-199 (05909; Nissui Pharmaceutical, Tokyo, Japan)
supplemented with 20% calf serum (12133C; Sigma-Aldrich, St.
Louis, MO, USA) was used as the basal solution. Dimethyl sulfoxide(DMSO; 13407-45, NACALAI TESQUE, Kyoto, Japan) and ethylene
glycol (EG; 15209-85, NACALAI TESQUE) were used as permeable

cryoprotectants. Sucrose (30404-45; NACALAI TESQUE) andcarboxylated poly-L-lysine (Bio Verde, Kyoto, Japan) were used as
nonpermeable cryoprotectants.
An equilibration solution consisting of 10% (v/v) DMSO and 10%(v/v) EG in basal solution and a vitriﬁcation solution containing 20%(v/v) DMSO, 20% (v/v) EG, 10% (w/v) carboxylated poly-L-lysine, and
0.5 M sucrose were prepared. A rewarming solution and a dilution
solution containing 1 M and 0.5 M sucrose, respectively, wereprepared, and the basal solution was used as the washing solution.
The vitriﬁcation solution was used at an ice-cold temperature (oncrushed ice). All other solutions were used at room temperature(24e27  C).
2.3. Vitriﬁcation and rewarming procedures
2.3.1. Circulating vitriﬁcation bag method
As a container for the vitriﬁcation of cell sheets, we developed
the circulating vitriﬁcation bag, which allows sequential ﬂushing ofsolutions during the pre- and post-vitriﬁcation processes. As shownin Fig. 1a, the circulating vitriﬁcation bag is a sealable polyethylene
bag to which inﬂow and outﬂow tubes for injecting or discharging
various solutions, respectively, are attached. The cell sheet was
sandwiched between two nylon meshes (45  45 mm, 95 mm
thread diameter, 140 mm opening diameter), and the four corners
were ﬁxed with a stapler. After inserting the cell sheet into thecirculating vitriﬁcation bag, the zipper was closed while removing
air in the bag (Fig. 1c1). The circulating vitriﬁcation bag was
attached to the stage of an orbital shaker (05-450-213; SANSYO Co.,
Ltd., Tokyo, Japan), and the solutions shown in Table 1 were injectedsequentially using a 20-ml syringe (Fig. 1b) while shaking the bagwith the following conditions; rotation number: 15 rpm, rocking
angle: 20 , turning angle: 360 , under room temperature(24e27  C). First, 10 ml equilibration solution-1 was injected via theinﬂow tube into the circulating vitriﬁcation bag (pre-equilibration;
Fig. 1c2). The cell sheet was kept in equilibration solution-1 for
5 min while shaking the bag (Fig. 1c3). Then, the solution wasdischarged from the outﬂow tube using a 20-ml syringe (Fig. 1c4).
By using this same procedure, the cell sheet was exposed toequilibration solution-2 for 12.5 min. After discharging the solution, vitriﬁcation solution-1 was injected while shaking the bag.
After conﬁrming the permeation of the solution into every part ofthe bag, the bag was removed from the shaker and placed on ice.
After exposure of the cell sheet to vitriﬁcation solution-1 for 5 min(pretreatment with vitriﬁcation solution), the bag was attached tothe shaker once again, and the solution was discharged. Based on
the same procedure described above, vitriﬁcation solution-2 wasinjected into the bag, and the cell sheet was exposed to the solution
on ice for 7.5 min. After the discharge of the solution, the circulating
vitriﬁcation bag was held 1 cm above the surface of liquid nitrogen(LN) in a 1-L Styrofoam container and vitriﬁed by exposure to theTable 1
Composition of each solution and the time and temperature of each step.
DMSO (v/v%)
Solutions for pre-vitriﬁcation treatment
Equilibration solution-1
10Equilibration solution-2
10Vitriﬁcation solution-1
20
Vitriﬁcation solution-2
20
Solutions for post-vitriﬁcation treatment
Rewarming solutioneDilution solutioneWashing solution-1eWashing solution-2e

EG (v/v%)

COOH-PLL (w/v%)

Sucrose (mol)Time (min)Temperature

10
10
20
20

e
e
10
10

e
e
0.5
0.5

5
12.5
5
7.5

RT
RT
IT
IT

e
e
e
e

e
e
e
e

1
0.5
e
e

1
3
5
5

RT
RT
RT
RT

COOH-PLL: Carboxylated poly-L-lysine; DMSO: Dimethyl sulfoxide; EG: Ethylene glycol; IT: Ice-cold temperature; RT: Room temperature.

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221

217

Fig. 1. The structure of the circulating vitriﬁcation bag and operation procedure. a: (Left) The structure of the circulating vitriﬁcation bag. An inﬂow tube and an outﬂow tube
(polytetraﬂuoroethylene tube, inner diameter: 1.2 mm) are attached to the polyethylene bag (110  85 mm; ﬁlm thickness: 0.063e0.064 mm). The inﬂow tube has 15 holes (holediameter: 0.6 mm) to allow quick inﬂow of the solutions (indicated by arrows). The outﬂow tube has 30 holes (hole diameter: 0.6 mm), allowing for smooth solution discharge(indicated by arrows). (Right) A photograph of the circulating vitriﬁcation bag. b: A circulating vitriﬁcation bag attached on an orbital shaker that was operated with the following
conditions; rotation number: 15 rpm, rocking angle: 20 , turning angle: 360 , under room temperature (24e27  C). c: Operation procedure of the circulating vitriﬁcation bag. (1)Insert the cell sheet sandwiched between two nylon meshes into the circulating vitriﬁcation bag and close the opening. Then, attach the bag to the shaker. (2) While shaking thecirculating vitriﬁcation bag, inject the equilibration solution-1 via the inﬂow tube (arrows). (3) Keep the cell sheet in solution for a given amount of time (Table 1). (4) Discharge thesolution in the circulating vitriﬁcation bag from the outﬂow tube (arrows). Repeat steps (2) to (4) to successively inject and discharge the pre- and post-vitriﬁcation solutions shownin Table 1.

vapor (approximately 150  C). After exposure to the LN vapor forat least 1 min, the cell sheet was rewarmed. The circulating vitriﬁcation bag was placed on a heating plate at 45  C. At the sametime, the bag was covered by a gel pack that had been preheated to
38  C for rapid rewarming. After 1 min, when the cell sheet hadcompletely rewarmed, the circulating vitriﬁcation bag was
attached again to the shaker. While shaking the circulating vitriﬁcation bag, the solutions for rewarming, dilution/removal of cryoprotectants, and washing (shown in Table 1) were successively
injected and discharged. The cell sheet was exposed to rewarming
solution for 1 min, dilution solution for 3 min, washing solution-1
for 5 min (prewash), and ﬁnally washing solution-2 for 5 min.
Following the washing process, the cell sheet sandwiched between
nylon meshes was removed from the bag using forceps and placed
into a 100-mm dish. The cell sheet was then retrieved from thenylon meshes using forceps and immersed in 5 ml of Caþþ- andMgþþ- free phosphate buffered saline (PBS()) in a 60-mm dish.
After checking for visible damage (cracks), the sheet was enzymetreated for the cellular viability assessment.
2.3.2. Envelope method
For comparison with the circulating vitriﬁcation bag method,the envelope method that we reported previously [4] was used tovitrify and rewarm chondrocyte sheets. Brieﬂy, a cell sheet was

successively immersed in 5 ml of the solutions used for pretreatment prior to vitriﬁcation (Table 1) in a 60-mm dish. The disheswith vitriﬁcation solution-1 and -2 were placed on ice. Whenpicking up the cell sheet using forceps, we treated the cell sheetwith extra care to avoid breaking it. After treating the cell sheetwith vitriﬁcation solution-2, it was placed on a 90  50 mm rectangular piece of aluminum foil (thickness: 12 mm; Mitsubishi
Aluminum Co., Ltd., Tokyo, Japan) using forceps. The aluminum foil
was folded to enclose the cell sheet [4] and was held 1 cm above theLN surface in a 1-L Styrofoam container, and the cell sheet was
vitriﬁed by exposure to vapor (approximately 150  C). After
exposure to LN vapor for more than 1 min, the cell sheet was
rewarmed.
For rapid rewarming, the cell sheet wrapped with aluminum foil
was placed on a heating plate at 38  C, and it was covered by a gel
pack that had been preheated to 38  C. One minute later, thealuminum foil was carefully unfolded, and the cell sheet was
retrieved using forceps without breaking it and successively
immersed in 5 ml of the solutions used for rewarming, dilution/
removal of cryoprotectants, and washing (Table 1) in a 60-mm dish.
After washing, the cell sheet was immersed in 5 ml of PBS() in a
60-mm dish, and after checking it for visible damage (cracks), it was
enzyme-treated for the cellular viability assessment.

218

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221

2.4. Evaluation of the cells composing the cell sheet

3. Results

The cell sheet viability assessment was conducted according to
the method used in our previous study [4]. Brieﬂy, the cell sheet
was cut into 1e2 mm2 pieces with ophthalmic scissors and incubated in DMEM/F12 medium containing 2 mg/ml collagenase II(17101; Thermo Fisher Scientiﬁc K.K.) at 37  C for 60 min to isolate
the component cells. The suspension of the isolated cells wascentrifuged at 1000 rpm for 5 min, and the precipitated cells were
resuspended in DMEM/F12 medium; the cell viability was determined after trypan blue staining [viability (%) ¼ live cells/live anddead cells  100].

3.1. Characteristics of post rewarming rabbit chondrocyte sheets
vitriﬁed with the circulating vitriﬁcation bag method

2.5. Histological examination and immunohistochemical staining
Histological examination and immunohistochemical staining
were performed according to our previous report [4]. Doublelayered cell sheets were harvested after culture or cryopreservation and ﬁxed in 4% paraformaldehyde solution for 1 week. The
specimens were embedded in parafﬁn, sectioned, and placed on
glass slides. After deparafﬁnization and rehydration, the sections
were stained for proteoglycan with 0.1% Safranin-O or immunostained with type II collagen antibody. For immunohistochemistry,
the slides were incubated with a diluted primary anti-human type
II collagen antibody (F-57; Daiichi Fine Chemical, Toyama, Japan)
for 16 h at 4  C, followed by incubation with the EnVision þ Mouse/
HRP secondary antibody (K4000; DAKO, Glostrup, Denmark) for
1 h at room temperature. Finally, the sections were stained withdiaminobenzidine (K3466; DAKO) and counterstained with hematoxylin. Coverslips were mounted onto slides and sealed with
nail polish. The slides were then examined under a microscope, andimages were captured (Biozero BZ-X710; KEYENCE, Osaka, Japan).
2.6. Experimental design
2.6.1. Veriﬁcation of the circulating vitriﬁcation bag method
To verify the performance of the newly developed circulating
vitriﬁcation bag method, a group of rabbit chondrocyte sheets werevitriﬁed using this new method and the previously reported envelope method. Fresh cell sheets were used as non-vitriﬁed controls. The sheet structure, viability of cells composing the sheet, anddistributions of proteoglycan and type II collagen, which are major
components of cartilage, were examined in vitriﬁed and nonvitriﬁed control sheets.
2.6.2. Long-term preservation of cell sheets using the vitriﬁcation
storage box
For the long-term storage and handling of vitriﬁed cell sheets,
which are vulnerable to physical damage due to devitriﬁcation, wedeveloped a vitriﬁcation storage box (Fig. 2aec). As shown in
Fig. 2b, the box is a thin stainless steel container with built-in LN
absorbents in the main body and the lid. In this experiment, weaimed to demonstrate that the vitriﬁcation storage box allows forlong-term storage of the rabbit chondrocyte sheet. The cell sheets
wrapped with aluminum foil following pretreatment with cryoprotectants were vitriﬁed in accordance with the envelope method[4] using the vitriﬁcation storage box. The vitriﬁcation storage box
containing the vitriﬁed sheet was transferred to and stored in LN
vapor (150  C) in an LN container (Fig. 2d) for a storage period of
one or six months. The cell sheets after the long-term storage periods were compared with the cell sheets rewarmed immediately
after vitriﬁcation (vitriﬁed controls) and fresh cell sheets (nonvitriﬁed controls) in terms of the cell sheet structure and cellularviability.

The circulating vitriﬁcation bag method was used to vitrify
rabbit chondrocyte sheets, and the integrity of the sheet structure
as well as the viability of the component cells were examined after
rewarming. All of the vitriﬁed sheets (n ¼ 7) showed no visible
damage and had the same appearance as non-vitriﬁed cell sheets(Fig. 3aec), and the average cell viability was 91.0 ± 2.9% (Table 2).
This cell survival rate was equal to that of the cell sheets vitriﬁed by
the envelope method (n ¼ 7; 89.5 ± 1.4%) and the non-vitriﬁed
control (n ¼ 7; 90.0 ± 3.0%).
In addition, portions of the cell sheets recovered after vitriﬁcation and rewarming were isolated to investigate the distributions ofproteoglycan and type II collagen using histochemical staining andimmunostaining. Similar to the cell sheet vitriﬁed with the envelope method and the non-vitriﬁed cell sheet, the cell sheets vitriﬁed with the circulating vitriﬁcation bag method showed strongsafranin-o staining throughout the tissue section (Fig. 3def).
These results indicated that proteoglycan was densely and evenly
distributed throughout the chondrocyte sheet. Moreover, the cellsheet also showed an even and abundant distribution of type II
collagen (Fig. 3gei). These data showed that the extracellular matrix of the cell sheets had been maintained after cryopreservation
with the circulating vitriﬁcation bag method.
3.2. Post rewarming structure and cellular viability of the cell sheet
stored for long periods using the vitriﬁcation storage box
The vitriﬁcation storage box was used to vitrify and store rabbit
chondrocyte sheets for long periods, and the sheet structure andcellular viability of post rewarming cell sheets were assessed(Table 3). There was no visible damage in the vitriﬁed cell sheets
stored for both 1 and 6 months in LN vapor in the LN container(Fig. 4). Furthermore, the rates of cell survival after storage for one
and six months (79.2 ± 2.6% and 81.2 ± 1.0%, respectively) wereequal to those of the cell sheets rewarmed immediately after
vitriﬁcation (80.1 ± 2.5%) and the non-vitriﬁed controls(84.3 ± 1.8%).
4. Discussion
Vitriﬁcation techniques permit cryopreservation of cells andtissues with a high concentration of cryoprotectants. The beneﬁt ofvitriﬁcation techniques is that the solution turns into a glassy(amorphous) state at ultralow temperatures without the formationof ice crystals that cause physical damage to the cells [6]. In thevitriﬁcation method, samples are exposed to high concentrations ofcryoprotectants. During the pretreatment process of cell sheet
vitriﬁcation, samples are exposed to various solutions to allow
cryoprotectants to permeate into the cells in a stepwise fashion atincreasing concentrations between the isotonic solution and thevitriﬁcation solution, which has an osmolarity of 11000 mOsmol.
Next, in the post-vitriﬁcation process, the cell sheet in ultrahypertonic vitriﬁcation solution is then exposed in a stepwise
fashion to solutions with decreasing concentrations, during whichcryoprotectants are diluted and eliminated. To complete theseprocesses, the previously reported vitriﬁcation method [4] required
a cumbersome, manual process to transfer the cell sheet successively from one solution to another; this method is unrealistic toimplement in actual clinical settings.
In contrast, the circulating vitriﬁcation bag method developed in
the current study allows the cell sheet to be exposed successively toA. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221

219

Fig. 2. The structure and operation procedure of the vitriﬁcation storage box. The vitriﬁcation storage box (a) and its internal structure (b). b: The main body of the vitriﬁcation
storage box (75  75  12 mm) and its lid (75  75  5 mm) are both made of stainless steel perforated plates (1 mm in thickness), and they are connected by a hinge. Both the main
body and the lid have a built-in liquid nitrogen (LN) absorbent. c: Three vitriﬁcation storage boxes can be accommodated in a stainless storage rack (76  78  57 mm). The storage
rack can be accommodated in an LN container canister (d-3). d: The operation procedure of the vitriﬁcation storage box. (1) Place the cell sheet wrapped in the vitriﬁcation packageon the perforated plate of the main body of the box that was immersed and cooled in LN. (2) Vitrify the cell sheet in the vitriﬁcation storage box by exposing it to LN vapor (150  C)
discharged from the LN absorbents. (3, 4) Place up to three vitriﬁcation storage boxes in a canister using the storage rack (3) and store them in LN vapor in the LN container (4). The
vitriﬁcation storage box was prepared on a special order by Umihira Co. Ltd., Kyoto, Japan.

various solutions with different components and osmolarity bykeeping the cell sheet in a sealed bag while successively replacing
the solution in the bag. The circulating vitriﬁcation bag methodminimizes the number of occasions the operator comes in contact
with the cell sheet, which is helpful in reducing labor andenhancing sterility when applying the cell sheet vitriﬁcation
technique in clinical settings.
The main concern in the application of this new method wasthat the residual solution in the preceding step within the bag may
not be completely replaced by the solution in the following step.
However, in our preliminary experiment, the solution in the bag
was conﬁrmed to be almost completely replaced by the new solution after just one round of preﬂushing (Suppl. Fig. 1). It can beinterpreted that, in the circulating vitriﬁcation bag method, the cellsheet was properly exposed to each solution in the vitrifyingrewarming process, which allowed it to achieve similar performances as a sheet treated by the conventional method that requires
manual handling of cell sheets.
In the cell sheet vitriﬁcation procedure, cells are exposed tolarge changes in the osmolarity of the solutions used. Rapidchanges in osmolarity can be a stress factor for cells [7]. However,the use of the circulating vitriﬁcation bag is thought to be beneﬁcial
for reducing the osmotic stress to cells during cell sheet treatment.
The reason for this is that in the circulating vitriﬁcation bag
method, the cell treatment solution used in the preceding step isgradually replaced by the solution in the subsequent step
(Suppl. Fig. 1). Therefore, the change in osmolarity during thisprocess is estimated to be milder. In other words, a well-designed
solution replacement method can minimize the stress due to osmotic changes while improving the survival rates of the component
cells of vitriﬁed cell sheets. As the bag used in this study was a
prototype, the optimization of both the inﬂow and outﬂow rates
will be required in the future.
This study found that the circulating vitriﬁcation bag method
was able to maintain the morphological integrity of the vitriﬁed
and rewarmed cell sheet. The survival rates of component cells andcomponents of the extracellular matrix were also maintained after
vitriﬁcation and were on par with those of the non-vitriﬁed sheets.
The recovery of cartilage damage using the chondrocyte sheet was
shown to involve bioactive substances secreted from the cell sheet,including transforming growth factor beta 1, melanoma inhibitory
activity, and prostaglandin E2 [5,8,9]. We have already conﬁrmed in

220

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221

Fig. 3. Comparative analysis of the characteristics of rabbit chondrocyte sheets before and after vitriﬁcation. Comparison of the morphological appearance (aec) of rabbit chondrocyte sheets and distribution of proteoglycan (def) and type II collagen (gei) in cross-sections of the cell sheets. a: There was no visible crack in the cell sheet vitriﬁed by thecirculating vitriﬁcation bag method. The extracellular matrix of the vitriﬁed cell sheet contained abundant proteoglycan (d) and type II collagen (g) with a dense and even distribution. These characteristics of the vitriﬁed cell sheets were similar to those of the cell sheets vitriﬁed by the envelope method (b, e, h) and non-vitriﬁed controls (c, f, i). (Scalebar ¼ 50 mm).

Table 2
Structural maintenance and cell viability after rewarming of rabbit chondrocyte sheets vitriﬁed by the circulating vitriﬁcation bag method.
Vitriﬁcation method

No. of cell sheets recovered without fracture/No. of cell sheets examined (%)

Cell viabilitya

Circulating vitriﬁcation bag method
Envelope method
Non-vitriﬁed control

7/7 (100)
7/7 (100)
7/7 (100)91.0 ± 2.9% (n ¼ 7)
89.5 ± 1.4% (n ¼ 7)
90.0 ± 3.0% (n ¼ 7)

aMean ± S. D.

Table 3Structural maintenance and cell viability of rabbit chondrocyte sheets preserved for long periods in the vitriﬁcation storage box.

Non-vitriﬁed control
Vitriﬁed controla
Vitriﬁed and storedab

Storage period

No. of cell sheets recovered without fracture/No. of cell sheets examined (%)

Cell viabilitybee
1 month
6 months

6/6 (100)
5/5 (100)
3/3 (100)
3/3 (100)

84.3 ± 1.8% (n ¼ 6)
80.1 ± 2.5% (n ¼ 5)79.2 ± 2.6% (n ¼ 3)
81.2 ± 1.0% (n ¼ 3)

Vitriﬁed control represents the cell sheet that was rewarmed immediately after vitriﬁcation.
Mean ± S. D.

Fig. 4. Morphological appearance of rabbit chondrocyte sheets after long-term vitriﬁcation preservation. The morphological appearance of rabbit chondrocyte sheets that werestored for long periods in the vitriﬁcation storage box was observed. a: Non-vitriﬁed control cell sheet. b: The cell sheet vitriﬁed with the envelope method and immediately
rewarmed (vitriﬁed control). c, d: Intact morphology of the cell sheets vitriﬁed in the vitriﬁcation storage box and stored in the LN container for one month (c) and six months (d).

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221

our previous study that these factors could be generated fromvitriﬁed and rewarmed rabbit chondrocyte sheets [5]. In the future,
we need to check whether the cell sheet vitriﬁed using the circulating vitriﬁcation bag method has the same cartilage damage recovery function.
In the present study, we did not test the applicability of thecirculating vitriﬁcation bag method to other cell sheet types than
rabbit chondrocytes. The vitriﬁcation protocol used in the circulating vitriﬁcation bag method has been proven to be effective forcryopreserving human myoblast cell sheet [10]. It is, therefore,
likely that the circulating vitriﬁcation bag method is applicable tovarious cell sheets.
In the development of vitriﬁcation methods for animal embryos,
including those of humans, the minimum volume cooling (MVC)
concept [11] has been widely used. By considering that the utility of
the MVC concept has been proven in embryo vitriﬁcation, wedeveloped the cell sheet vitriﬁcation method based on this concept[4]. In methods based on the MVC concept, the sample is cryopreserved along with a minimum volume of vitriﬁcation solution.
By minimizing the amount of vitriﬁcation solution used, the speedof the temperature decrease or increase of the sample is maximized. During the temperature fall and rise, the solution quickly
passes through the temperature range at which ice crystals are
more likely to form. In this way, the risk of ice crystal formation in
the solution is reduced [12], and a high sample survival rate can be
maintained.
On the other hand, one drawback of methods based on the MVC
concept is that they are more likely to result in devitriﬁcation. A cellsheet vitriﬁed with a small amount of vitriﬁcation solution devitriﬁes even with a slight rise in temperature. Devitriﬁcation under
non-optimal conditions may result in cell damage associated withice crystal formation in solution. Such devitriﬁcation tends to occur
when pulling the vitriﬁed cell sheet out of the LN container ortransferring it within the laboratory or between facilities. Here, wedeveloped a vitriﬁcation storage box that is effective in the prevention of devitriﬁcation in the abovementioned situations.
Moreover, the vitriﬁcation storage box can be used in the entire
process, from vitriﬁcation of the cell sheet to storage and transportation. The characteristics of the cell sheet vitriﬁcation methodthat we previously developed [4] allow it to induce vitriﬁcation by
exposing the sample to LN vapor (approximately 150  C) rather
than by immersing it in LN. The internal temperature of the vitriﬁcation storage box ﬁlled with LN absorbents is maintained stably
at approximately 150  C, which produces the desired vitriﬁcation
conditions and preservation conditions of the vitriﬁed cell sheet.

5. Conclusions
In conclusion, the results of this study indicated that the circulating vitriﬁcation bag method was potentially capable of realizing
the clinical application of vitriﬁed chondrocyte sheets. The vitriﬁcation storage box was also shown to be useful for the long-term

221

preservation of vitriﬁed cell sheets, making the clinical application of cryopreserved chondrocyte sheets even more feasible.
Declaration of Competing Interest
Asuka Hayashi, Miki Maehara, Ayuko Uchikura and Hitomi
Matsunari declare that they have no conﬂict of interest. Kazuaki
Matsumura and Suong-Hyu Hyon are cofounders of Bioverde Inc.
Masato Sato receives research funding from CellSeed Inc. Hiroshi
Nagashima is a founder and shareholder of PorMedTec Co., Ltd.
Acknowledgments
This work was supported by the Meiji University International
Institute for Bio-Resource Research (MUIIBR), and the Research
Project for Practical Applications of Regenerative Medicine (No.
JP19bk0104063 to MS) from the Japan Agency for Medical Researchand Development. The authors thank Mr. Y. Tokuyama for his
technical assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online athttps://doi.org/10.1016/j.reth.2020.04.006.
References[1] Sato M, Yamato M, Mitani G, Takagaki T, Hamahashi K, Nakamura Y, et al.
Combined surgery and chondrocyte cell-sheet transplantation improvesclinical and structural outcomes in knee osteoarthritis. npj Regen Med 2019;4:
4.
[2] Toyoda E, Sato M, Takahashi T, Maehara M, Okada E, Wasai S, et al. Transcriptomic and proteomic analyses reveal the potential mode of action ofchondrocyte sheets in hyaline cartilage regeneration. Int J Mol Sci 2019;21(1).
[3] Maehara M, Sato M, Toyoda E, Takahashi T, Okada E, Kotoku T, et al. Characterization of polydactyly-derived chondrocyte sheets versus adult chondrocyte sheets for articular cartilage repair. Inﬂamm Regen 2017;37:22.
[4] Maehara M, Sato M, Watanabe M, Matsunari H, Kokubo M, Kanai T, et al.
Development of a novel vitriﬁcation method for chondrocyte sheets. BMC
Biotechnol 2013;13:58.
[5] Tani Y, Sato M, Maehara M, Nagashima H, Yokoyama M, Yokoyama M, et al.
The effects of using vitriﬁed chondrocyte sheets on pain alleviation andarticular cartilage repair. J Tissue Eng Regen Med 2017;11:3437e44.
[6] Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196 degrees C by vitriﬁcation. Nature 1985;313:573e5.
[7] Matsumura K, Hyon SH. Polyampholytes as low toxic efﬁcient cryoprotective
agents with antifreeze protein properties. Biomaterials 2009;30:4842e9.
[8] Kaneshiro N, Sato M, Ishihara M, Mitani G, Sakai H, Mochida J. Bioengineeredchondrocyte sheets may be potentially useful for the treatment of partial
thickness defects of articular cartilage. Biochem Biophys Res Commun
2006;349:723e31.
[9] Hamahashi K, Sato M, Yamato M, Kokubo M, Mitani G, Ito S, et al. Studies of
the humoral factors produced by layered chondrocyte sheets. J Tissue Eng
Regen Med 2015;9:24e30.
[10] Ohkawara H, Miyagawa S, Fukushima S, Yajima S, Saito A, Nagashima H, et al.
Development of a vitriﬁcation method for preserving human myoblast cellsheets for myocardial regeneration therapy. BMC Biotechnol 2018;18(1):56.
[11] Hamawaki A, Kuwayama M, Hamano S. Minimum volume cooling method forbovine blastocyst vitriﬁcation. Theriogenology 1999;51(1):165.
[12] Wowk B. Thermodynamic aspects of vitriﬁcation. Cryobiology 2010;60:
11e22.