Using a Novel Supramolecular Gel Cryopreservation System in

Subscriber access provided by UNIV OF DURHAM

Interface-Rich Materials and Assemblies

Using a Novel Supramolecular Gel Cryopreservation System in Microchannel to Minimize the Cell Injury Dong xu Lan, Xi Chen, Pengcheng Li, Wei Zou, Lili Wu, and Wanyu Chen Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00265 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Using a Novel Supramolecular Gel Cryopreservation System in Microchannel to Minimize the Cell Injury Dongxu Lan†, ‡, Xi Chen †, ‡, Pengcheng Li†, Wei Zou†, Lili Wu†, Wanyu Chen*† *: Corresponding Author †: School of Materials Science and Engineering, Wuhan University of Technology, Wuhan, Hubei 430070, China KEYWORDS: Gelator, Supramolecular gel, Cryopreservation, Cell injury. ABSTRACT: The storage of living cells is the major challenge for cell research and cell treatment. Here, we introduced a novel supramolecular gel cryopreservation system which was prepared in microchannel and the supramolecular gel (BDTC) was self-assembled by gelator Boc-O-dodecyl-L-tyrosine (BDT). This cryopreservation system could obviously minimize the cell injury because BDTC supramolecular gel had a more compact three-dimensional network structure when BDT gelator self-assembled in the confined space of microchannel. This compact structure could confine the growth of the ice crystal, reduce the change rate of cell volumes and osmotic shock, decrease the freezing point of the cryopreservation system and possess better protection capability. Furthermore, the results of functionality assessments showed that the thawed cells could grow and proliferate well and remain the same growth trend of the fresh cells after the RSC96 cells flowed out from the 1

ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 45

microchannel. This novel method has potential to be used for the cryopreservation of cells, cell therapy and tissue engineering.

INTRODUCTION Cells are a necessary part of cell transplantation and biological organ research.

[1-2]

In

recent years, with the development of biological technology, a large number of high activity cells is paid more attention by researchers and the long-term storage of cells is a huge challenge for us to face. At present, the main storage method is cryopreservation which is a technique for the storage of cells, tissues or other materials at low temperature.

[3-4]

Generally, the living cells are cooled to low

temperature by special methods. The metabolism in the cell is inhibited by the low temperature so that the cells can be stored for a long time. When the cells are needed to be used, the cryopreservation system can be thawed to the physiological temperature (37 °C) in a special way, and the living cells can be obtained. In recent years, cryopreservation is of highly significance in many biological fields, such as the modern medicine and the cryobiology. [5-6] Usually, cryoprotectants (CPAs) are added to cryopreservation system because they can protect cells or tissues during the process of freezing and thawing. CPAs include permeable cryoprotectants (PCs) and impermeable cryoprotectants (IPCs).

[7]

PCs can penetrate into the cell and they are

mainly small molecules such as dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerin, propanediol (PG), etc. IPCs are not able to penetrate into the cells because they are usually large molecules and low-molecular saccharides like sucrose, 2


Page 3 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

trehalose, amino acid, polymer ethylene glycol (PEG), etc.

[8]

For example, Gao’s

group used 1.5 M dimethyl sulfoxide (DMSO) in CPTES solution (a balanced salt solution similar in composition to intracellular fluid, and buffered with 100 mM N-tris(hydroxylmethyl) methyl-2-aminoethanesulfonic acid) as a cryoprotectant to cryopreserve the rabbit carotid arteries and they found that the cryopreserved arteries still retained a relatively high patency rate after 1 year.

[9]

But in the actual storage

process, because of the formation and growth of the intracellular ice crystal and the extracellular ice crystal, the drastic alteration of osmotic pressure, the recrystallization in the process of thawing and the toxicity of CPAs, the traditional cryopreservation will destroy the cellular structure, induce the cell dehydration and lead to cellular osmotic injury. [10-11] In recent years, the hydrogels have been wildly used in many fields such as clinical medicine, cell culture and cell cryopreservation because they have good biocompatibility and can entrap the cells.

[12-14]

Alginate is the most commonly used

material to form hydrogels because it is biocompatible and has suitable permeability. Paulraj Kanmani and coworkers used the alginate-chitosan gel to encapsulate probiotic cells and the results showed better survival of streptococcus phocae cells (5.468 ± 0.15 Log CFU/mL) with high bacteriocin activity at −20 °C for six months because of the protection of the three-dimensional network structure of the gels.

[15]

Wanyu Chen and coworkers used the alginate hydrogel microcapsules containing fluorescent oxygen-sensitive dye to encapsulate the single rat pancreatic islets and these microcapsules could test the effect of cryopreservation on the oxygen 3


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

consumption rate (OCR) of single islets.

[16]

Page 4 of 45

Although the hydrogels have many

advantages to be used for cryopreservation, the biggest problem is that the hydrogel membrane is hard to be removed without damaging the cells, for example the membrane is removed by adding ethylene diamine tetraacetic acid (EDTA), trisodium citrate or other reagents. [17] Supramolecular gels are self-assembled by gelators which are low-molecular-weight compounds and the driving forces of self-assembly are intermolecular forces such as hydrogen bonding and van der Waals.

[18-19]

They are reversible and sensitive to the

change of the environment because the noncovalent interactions have reversibility. Moreover, they usually have three-dimensional network structures and they are biocompatible and biodegradable. Thus, the supramolecular gels have been widely used in biomedicine, cell engineering and drug delivery.

[20-21]

However, there are

rarely researches about using supramolecular gels in the field of cryopreservation. Jie Zeng and his coworkers found the supramolecular gel could minimize neural cells injury during cryopreservation process because the supramolecular gel could reduce the permeation rate of CPAs, decrease the change rate of osmotic pressure and the ice injury.

[22]

In addition, the supramolecular gel could turn into liquid and be removed

easily in the process of thawing. Microfluidics, which emerged at the beginning of the 1980s and are wildly used in many fields discovery

[23-24]

[29-30]

such as biological preservation [25-26], cell diagnosis

[27-28]

, and drug

in recent years. Compared with the traditional analysis tools, the

microfluidics technology has many advantages as follows：Firstly, it can flexibly and 4


Page 5 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

accurately control the fluid whose volume is nanolitre or picoliter in the microchannel. Secondly, the microfluidics technology possesses faster analysis speed, higher detection sensitivity and less samples and reagents. In addition, because the microchannel has suitable width and size to the cells, a micro environment for cells in vitro can be created. Xu Y and coworkers encapsulated the mouse fibroblast cell line L929 cells and the human aortic endothelial cells (HAECs) in the hydrogel via microfluidics technology and the viability of HAECs was 93.9% after 7-day preservation, the viability of L929 cells was 92.1% after 8-day preservation.

[31]

Although there is no clinical application of microfluidic technology in cell cryopreservation, the related research reports are also increasing with the further popularization of microfluidic technology.

[32-34]

Yujie Zou and coworkers used

microchannel to accomplish the ultra-rapid cryopreservation of the human spermatozoa without cryoprotectant and the human spermatozoa’s physiological parameters were almost not influenced by cryopreservation. [35] In addition, microchannel can tune the network structures of supramolecular gels by utilizing geometric confinement.

[36]

However, to our knowledge, the supramolecular

gels in microchannel are almost not used in the field of cryopreservation. Hence, in this paper, we introduced a novel supramolecular gel (BDTC) cell cryopreservation system in microchannel to minimize the cell injury. RSC96 cells were used as model cell and we found that the viability of RSC96 cells was increased because the use of microchannel could tune the microstructure of BDTC supramolecular gel to be more

5


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

compact. And the multicomponent cryoprotectant system had better protective effect than single component cryoprotectant system. EXPERIMENTAL SECTION Preparation of Gelator Boc-L-tyrosine methyl ester, N,N-dimethylformamide (DMF), 1-bromododecane (analytical grade) were purchased from Sigma-Aldrich, China. The gelator was prepared as described in reference 37.

[37]

Briefly, as shown in Figure 1, 3.998 g

(0.0135 mol) Boc-L-tyrosine methyl ester was dissolved in 10ml DMF. Then 3.7113 g (0.0270 mol) potassium carbonate was added as acid-binding agent and 4ml (0.0167 mol) 1-bromododecane was added. This reaction lasted for 12 hours at room temperature. Finally, Boc-O-dodecyl-L-tyrosine methyl ester (BDTE) was obtained after recrystallization by absolute ethyl alcohol. 0.5007 g (0.0011 mol) BDTE was dissolved in 10 ml ethyl alcohol, 1.7 ml (0.0017 mol, 1 mol/L) sodium hydroxide solution was added into the mixture dropwise at the ice water bath. The reaction lasted for 1 hour at 0°C and then 5 hours at room temperature. The hydrochloric acid (0.2 mol/L) was added dropwise into the reaction system with vigorous stirring until the solution’s pH value reached 1-2. After filtering and drying for 24 hours in vacuum drying oven, 0.4858g Boc-O-dodecyl-L-tyrosine (BDT) was obtained.

6


Page 6 of 45

Page 7 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 1.The synthesis of gelator BDT.

Preparation of Microchannel We used microfluidic devices to cryopreserve cells and the microfluidic devices were fabricated by soft lithography techniques. The polydimethylsiloxane (PDMS) prepolymer and its curing agent (from Dow Corning Corporation, USA) were proportionally mixed (weight ratio of 10:1). After being stirred for 5 minutes, the mixture was poured on the silicon wafer (from New Way Photomask Making Co. LTD, China) with a microchannel pattern. After the vacuum pumping, the microchannel was heated for 3 hours at the temperature of 60 °C until the PDMS was cured completely. Then the cured PDMS was separated from the silicon wafer. The inlets and outlets of the microchannel were opened. The microchannel and the clean glass slide were disposed by plasma surface processor. After this, the glass slide was glued rapidly with the PDMS engraved with the pattern. The well bonded PDMS and

7


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

glass slides were cured for 2-3 hours at 60 °C, then the microchannel was accomplished. Morphology Analysis of Supramolecular Gel In ordinary group, the RPMI 1640 medium with BDT (3 g/L) was dropped on the glass slide at room temperature and the slide was placed at 4 °C or -80 °C for half an hour to conduct the gelation. In microchannel group, the RPMI 1640 medium with BDT (3 g/L) was injected into the microchannel at room temperature. Then the microchannel was placed at 4 °C or -80 °C for half an hour to conduct the gelation. The morphology of the BDTC supramolecular gel was observed by optical microscope (OLYMPUS-IX71, Japan). Thermal Analysis of Supramolecular Gel The BDTC supramolecular gel samples were analyzed by differential scanning calorimeter (DSC; PYRIS1DSC, from PerkinElmer, America). In the control group, DMSO (8 vol%) was added in RPMI 1640 medium. In the experiment group A, DMSO (8 vol%) and BDT(0.75 g/L) were added in RPMI 1640 medium. In the experiment group B, DMSO (8 vol%), BDT(0.75 g/L) and ethylene glycol (6 vol%) were added in RPMI 1640 medium. In the experiment group C, DMSO (8 vol%) and BDT(0.75 g/L) and trehalose (0.05 mol/L) were added in RPMI 1640 medium. The temperature was decreased from 40 °C to -80 °C with a cooling rate of 2 °C/min in the process of analysis.

8


Page 8 of 45

Page 9 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

The Cell Cryopreservation in Cryovial BDT gelator (1.5 g/L) was dissolved in the RPMI 1640 medium with 10 vol% fetal bovine serum (FBS) (from Thermo Fisher Scientific, China) and 1 vol% double-antibody (from Gibco, USA) in sonic oscillator at about 60 °C. After BDT completely dissolved, the mixture was filtered by using millipore filter (the pore size is 0.22 µm) to sterilize. After filtration, RSC96 cells were added into the mixture and the concentration of cell suspension was adjusted to 2×106 cells/mL. 0.5 ml RSC96 cell suspension was added into the 2 mL cryovials (Fisher Scientific, Pittsburgh, PA). Then the cryovials were put into 0~4 °C ice/water bath. Subsequently, the equivoluminal culture medium with CPAs, such as DMSO, ethylene glycol and trehalose (from Sigma-Aldrich, China) were added dropwise to the RSC96 cell suspension. The details of CPAs were shown in Table 1. The cryovials were put into a freezing container (from Nalgene Mr. Frosty Cryo 1 °C, Thermo Fisher Scientific Inc.) at the cooling rate of about 1 °C/min, and then the freezing container was placed in -80 °C freezer. After storage for 7 days, the cryovials were thawed in 37 °C water bath for about 2 minutes. Then the RSC96 cell suspension was centrifuged at 1000 r/min for 15 minutes to remove the CPAs and BDT. The centrifugal process was repeated 2~3 times. After thawing, the RSC96 cells were inoculated into 96-well plates with culture medium and cultured in the incubator (37 °C, 5% CO2). The operations above were conducted in the biological fume hood to ensure a strict aseptic environment.

9


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Cell Cryopreservation in Microchannel The RSC96 cell suspension (2×106 cells/mL) with BDT gelator was prepared as mentioned above. The process of cell cryopreservation in the microchannel was shown in Figure 2A. The polyethylene tubing (0.38 mm i.d., 1.09 mm o.d., from Becton Dickinson and Company, USA) was used to connect the microchannel entrances with the syringes. The RSC96 cell suspension containing BDT gelator and CPAs were injected to the microchannel by the micro flow injection pump (from NE-1000, Farmingdale, NY, USA) at the flow rate of 10 µL/min. And then the microchannel devices were placed in the Box-in-Box (BIB) system which is a custom-designed freezing container. According to reference 16, the BIB system was a reliable passive cooling rate-controlled device which could provide the cooling rate at about 1 °C/min. Then the freezing container was placed in a constant temperature refrigerator at -80 °C.

Figure 2. The process of cell cryopreservation and thawing in the microchannel. A: The process of cell cryopreservation. B: The process of cell thawing. 10


Page 10 of 45

Page 11 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

The process of cell thawing in the microchannel was shown in Figure 2B. After the storage for 7 days, the microchannel was placed in a constant temperature water bath (DK-98-II, Taisite, China) at 37 °C, and the supramolecular gel system turned into liquid state. After thawing, the culture medium was injected into the microchannel. Then the RSC96 cells were washed out (4~5 times) by the RPMI 1640 medium and were collected by the centrifuge tube at the export of the microchannel. The washing liquid was centrifuged for 15 minutes at a speed of 1000 r/min and then the supernatant was discarded. The centrifugal process was repeated 2~3 times. Then the RSC96 cells were placed to the 96-well plates and the culture medium of RPMI 1640 was added. The RSC96 cells were placed in the incubator (37 °C, 5% CO2). The operations above were conducted in a clean work station to ensure sterility. Trypan Blue Exclusion Assay The viability of the thawed RSC96 cells was assessed by using the trypan blue exclusion assay. The experimental groups were shown in Table 1. Table1. The experimental groups Group

Components

Storage Space

CB

10 vol% DMSO

cryogenic vials

MB

10 vol% DMSO

microchannel

MLB

6 vol% DMSO

microchannel

CG

6 vol% DMSO, 0.75 g/L BDT

cryogenic vials

MG


microchannel

MGE

10 vol% DMSO, 0.75 g/L BDT, 6 vol% ethylene glycol

microchannel

11


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 45

MGT

10 vol% DMSO, 0.75 g/L BDT, 0.05 mol/L trehalose

microchannel

MLG


microchannel

MLGE

6 vol% DMSO, 0.75 g/L BDT, 6 vol% ethylene glycol

microchannel

MLGT

6 vol% DMSO, 0.75 g/L BDT, 0.05 mol/L trehalose

microchannel

After thawing, the RSC96 cell suspension was taken out and centrifuged for 15 minutes (set at a speed of 1000 r/min) and then the supernatant was discarded. The density of cell suspension was adjusted to 1×106 cells/ml. Nine drops of the RSC96 cell suspension and one drop of the 0.4% trypan blue solution (4% trypan blue solution was diluted by PBS buffer solution) were added into a small test tube. The mixture was observed under the fluorescence microscope in 3 minutes. Necrotic cells were stained light blue, while the living cells were not stained. Then the living cells and necrotic cells were counted by hemocytometer. Fluorescent Staining Assay Some Hoechst 33342 could penetrate into the cell membrane of normal cells so the normal cells showed light blue fluorescence. More Hoechst 33342 could penetrate into the cell membrane of the apoptotic cells so the apoptotic cells showed bright blue fluorescence. In addition, PI could penetrate into the cell membrane of the necrotic cells but could not enter the normal cell so the necrotic cells showed red fluorescence. So after the thawed RSC96 cells were cultured for 3 days, they were stained by Hoechst 33342 and propidium iodide (PI, from Beyotime Biotechnology, China) double staining kit and the viabilities of the RSC96 cells were assessed by the fluorescent staining method. The processes were as follows: after thawing, the RSC96 12


Page 13 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

cell suspension was taken out and centrifuged for 15 minutes (set at a speed of 1000 r/min) and then the supernatant was discarded. The density of cell suspension was adjusted to 6×103 cells/ml. Then the RSC96 cell suspension was injected to 96-well plates (100 µl per hole). The 96-well plates were placed in culture incubator (37 °C, 5% CO2) for 3 days. Then the supernatant in the 96-well plates was sucked out. The 96-well plates were washed with PBS for twice. Then the prepared staining solution (200 µl) which was composed of Hoechst 33342, PI and Cell Staining Buffer (volume ratio1:1:200) was injected into the holes. The 96-well plates were placed in 4 °C for 20~30 minutes. Then it was observed under the fluorescence microscope. The groups of the experiment were the same as the trypan blue exclusion assay. MTT Assay The

degree

of

RSC96

cells

viabilities

was

tested

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

by

a

bromide

colorimetric (MTT)

colorimetric assay. RSC96 cell suspensions (100 µL per hole) were inoculated to 96-well plates at the concentration of 6×103 cells/mL and cultured in incubator (37 °C, 5% CO2). When the RSC96 cells were cultured for 0.5 day, 20 µL MTT solution (5 mg/mL) was added to each well and cultured for 4 hours in incubator (37 °C, 5% CO2). Then the same steps were repeated after 1 day, 2 days, 3 days and 5 days, respectively. Subsequently, the supernatant was sucked out, then 150 µL DMSO was added to each well and shook strongly for 10 minutes to dissolve formazan thoroughly. Samples were measured by spectrophotometric method at 570 nm by an

13


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

enzyme-linked immunosorbent assay plate reader (Type 1510, Thermo Fisher Scientific, USA). RESULTS AND DISCUSSION The results and discussions are as follows. All the experiments were repeated 6~8 times. The statistical analysis was performed using the two-tailed Student’s t--test and the P-value less than 0.05 was considered statistically significant. The Morphology of the BDTC Supramolecular Gel The morphology of the BDTC supramolecular gel was shown in Figure 3. The Figure 3A and Figure 3C showed the morphology of BDTC supramolecular gel samples (self-assembled by 1.5 g/L BDT) on the glass slides at 4 °C and -80 °C, respectively. The gelation temperature of the BDTC supramolecular gel was 8.36 °C which was shown in supporting information. When the temperature was decreased to 4 °C, the BDT gelator self-assembled to fibril-like aggregates via noncovalent forces. The fibres formed a tree-like microstructure at the beginning, then they intertwined and the compacted three-dimensional network structure with small pores was formed finally. The water in the supramolecular gels would crystallize in these small pores so the size of the ice crystal would be confined by the three-dimensional network structure of the supramolecular gels. The Figure 3B and Figure 3D showed the morphology of BDTC supramolecular gel samples (self-assembled by 1.5 g/L BDT) in the microchannel at 4 °C and -80 °C, respectively. The microstructure of BDTC supramolecular gel in microchannel was different to the microstructure of BDTC supramolecular gel on the slide glass. As 14


Page 14 of 45

Page 15 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

reported in the reference 36, when the concentration of the gelator in the confined space was the same to the unconfined space, the compact three-dimensional network structure was easier to be formed in the confined space. When the fibres grew and approached the side wall of microchannel, the growth would end or change the directions. Then the fibres would cross each other and twine to form a more compact network structure in the microchannel. Our results were similar. As shown in Figure 3B and Figure 3D, the BDTC supramolecular gels which were self-assembled in microchannel had more compact structure than the supramolecular gels which were self-assembled on the slide glass.

Figure 3. The morphology of the BDTC supramolecular gel: (A) The supramolecular gel was self-assembled on the slide glass at 4 °C. (B) The supramolecular gel was self-assembled in the microchannel at 4 °C. (C) The supramolecular gel was self-assembled on the slide glass at -80 °C. (D) The supramolecular gel was self-assembled in the microchannel at -80 °C.

15


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Thermal Analysis of Supramolecular Gel The DSC assay was used to analyse the enthalpy change of the BDTC supramolecular gel in cryopreservation. The final results were shown in Figure 4.

Figure 4. The DSC thermograms. Control Group: DMSO (8 vol%) was added in RPMI 1640 medium. Experiment Group A: DMSO (8 vol%) and BDT(0.75 g/L) were added in RPMI 1640 medium. Experiment Group B: DMSO (8 vol%), BDT(0.75 g/L) and ethylene glycol (6 vol%) were added in RPMI 1640 medium. Experiment Group C: DMSO (8 vol%), BDT(0.75 g/L) and trehalose (0.05 mol/L) were added in RPMI 1640 medium. As shown in Figure 4, the freezing point of the control group, the experimental group A, the experimental group B and the experimental group C were -16.8 °C, -19.1 °C, -25.7 °C and -20.2 °C, respectively. Based on the research results of Jie Zeng and coworkers,

[22]

the states of water can be divided into freezing free water, freezing

bonded water, and non-freezing water. The freezing point of freezing free water is the same as the pure water. The freezing bonded water can crystallize in the 16


Page 16 of 45

Page 17 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

cryopreservation process but the freezing point will decrease. And the non-freezing water cannot crystallize because of the strong interactions between the supramolecular gel networks and water. The three-dimensional network microstructure of supramolecular gels can reduce the freezing free water content and increase the freezing bonded water content. Thus, the formation of supramolecular gels can not only confine the growth of ice crystal but also decrease the freezing point of the whole cryopreservation system. In addition, the cryoprotection mechanism of ethylene glycol has been reported in the references. [38-39] Each ethylene glycol molecule has two hydroxyls and is easy to form hydrogen bond with the water molecule. The content of crystal water is reduced and the growth of the ice crystal is confined so that the freezing point of the whole cryopreservation system is decreased. Moreover, the trehalose molecule also can form hydrogen bond with the water molecule so trehalose can decrease the freezing point of water. But the hydrogen-bond interaction between the trehalose and water is less than it between the ethylene glycol and water.

[39]

So the freezing point of Group B (6 vol% ethylene

glycol, -25.7 °C) was lower than Group C (0.05 mol/L trehalose, -20.2 °C). Cell Viability The RSC96 cell viability which was detected by the trypan blue staining assay was shown in Figure 5A. The RSC96 cell viability which was detected by Hoechst 33342 and PI double staining kit was shown in Figure 5B. In addition, the microscopic

17


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

morphology of cells under fluorescence microscope was shown in supporting information.

Figure 5. The results of the RSC96 cells viabilities. A: The thawed RSC96 cells were detected by the trypan blue staining assay. B: The thawed RSC96 cells were detected by Hoechst 33342 and PI double staining kit. The experimental groups (CB, MB, CG, MG, MGE, MGT, MLB, MLG, MLGE, MLGT) were shown in Table.1 As shown in Figure 5, the results of trypan blue exclusion assay and fluorescent staining assay showed the cell viability of CB were 67.5 ± 2.35% and 62.5 ± 1.35%, 18


Page 18 of 45

Page 19 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

respectively. The results of trypan blue exclusion assay and fluorescent staining assay showed the cell viability of CG were 73.6 ± 3.32% and 71.3 ± 3.54%, respectively. As well as CB group and CG group, the cell viability was increased after the BDT was added when the other conditions were the same. For example: the results of trypan blue exclusion assay showed the cell viability increased from 68.1 ± 2.65% (MB) to 77.8 ± 4.26% (MG) and the results of fluorescent staining assay showed the cell viability increased from 63.7 ± 1.68% (MB) to 75.6 ± 4.02% (MG). At present, the main cell injuries in the cryopreservation are as follows: (1) ice injury: the cell injury is caused by the formation of intracellular and extracellular ice crystals during the cooling process; (2) solution injury: in the early stage of the cooling, the concentration of extracellular solution increases because the extracellular water crystallizes. Subsequently, the increase of the extracellular solution osmotic pressure leads to a large amount of water oozes out of the cells and the concentration of the intracellular solution increases, so the cells are injured by the highly concentrated electrolytes and solutes; (3) the cell injury is caused by the recrystallization of ice crystals during thawing process; (4) the cell injury is caused by osmotic pressure and cell volume excursion during the adding and removing of CPAs; (5) the cell injury is caused by the cytotoxicity of CPAs when the cells are exposed to the CPAs for a long time at room temperature. During the cooling process, because the temperature was lower than the gelation temperature of BDTC supramolecular gel medium system, the gelator BDT could self-assemble to a three-dimensional porous network structure by hydrogen bonding. The BDTC supramolecular gels served as the protective 19


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 45

membranes of the cells. The porous network structure enabled the BDTC supramolecular gels to encapsulate the culture medium in their pores of which the sizes were from 2 µm to 7 µm as shown in Figure 3A. The growth of the extracellular ice crystals was confined in these pores as shown in Figure 3C in the freezing process and the freezing point of cell cryopreservation system was reduced as shown in Figure 4. Therefore, when the gelator BDT was added, the BDTC supramolecular gel membrane could encapsulate the cryopreserved cells and effectively reduced the ice injury to the cells during the cryopreservation. Moreover，Figure 5 showed when the cells were cryopreserved in microchannel, the cell viability was increased, such as: the results of trypan blue exclusion assay and fluorescent staining assay showed the cell viability of CG were 73.6 ± 3.22% and 71.3 ± 3.54% and the cell viability of MG were 77.8 ± 4.26% and 75.6 ± 4.02%, respectively. In the gel cryopreservation system, there is a layer of gel membrane on the surface of the cell and the permeation process of the PCs follows the K-K model (as shown in formula (1)-(3)). [22, 32] (1)

‫ܬ‬௪ = ‫ܮ‬௉ ∆ܲ − ‫ܮ‬௉ ߪܴܶ∆ܿ ܲ௜ − ܲ௘ = ‫ܧ‬ ௗ௏ೢ ௗ௧

௏ି௏బ ௏బ

+ ܲ଴௜ − ܲ଴௘

(2) (3)

= ‫ܬ‬௪ ‫ܣ‬

‫ܬ‬௪ —Infiltration capacity ߪ—Cell membrane reflection coefficient of the cryoprotectant ‫ܮ‬௉ —Membrane hydraulic conductivity 20


Page 21 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

∆ܿ —Concentration

difference

of

cryoprotectant

between

intracellular

and

extracellular

‫— ܧ‬Cellular elastic modulus ‫—ܣ‬Cell membrane surface area i and e refer to intracellular and extracellular regions. In the BDTC supramolecular gel system, CPAs and water permeated into the gel membrane before they permeated into RSC96 cells since there was a layer of gel membrane on the surface of the cell. So the membrane hydraulic conductivity (‫ܮ‬௉ ) was decreased in the BDTC supramolecular gel cryopreservation system. And as shown in Figure 3, compared with the structure which was formed in the unconfined space such as the cryogenic vial, the fibres of BDTC supramolecular gel grew along the axial direction of the microchannel and formed an orderly, intertwined and more compact three-dimensional network structure when BDT gelator self-assembled in microchannel.

[36, 40]

Thus, on the one hand, the more compact network structure and

smaller pores could be easier to inhibit the formation of extracellular ice crystal in microchannel. On the other hand, ‫ܮ‬௉ was decreased again because of the more compact network structure of BDTC supramolecular gel which was self-assembled in the microchannel. Then the infiltration capacity (‫ܬ‬௪ ) and the cell volume change rate (

ௗ௏ೢ ௗ௧

) were decreased. Compared with the cryogenic vial, when the microchannel

was used as a container in the freezing process, the ice injury and osmotic injury were decreased. Therefore, the BDTC supramolecular gel could obtain better protection capability. 21


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In addition, when the concentration of DMSO was decreased from 10% to 6% in the microchannel, the results of trypan blue exclusion assay showed the cell viability became 72.9 ± 3.22% (MLG) and the results of fluorescent staining assay showed the cell viability became 72.1 ± 3.66% (MLG). These results were almost equal to the thawed cell viability of common cryopreservation in which the results of trypan blue exclusion assay and fluorescent staining assay showed the cell viability became 73.6 ± 3.22% (CG) and 71.3 ± 3.54% (CG), respectively. Thus, the results showed that the use of the microchannel in the process of cryopreservation could effectively reduce the concentration of the CPAs and decrease the toxicity of CPAs in cryopreservation system. As shown in Figure 5, the results of trypan blue exclusion assay and fluorescent staining assay showed the cell viability of MG were 77.8±4.26% and 75.6±4.02%, respectively. The results of trypan blue exclusion assay and fluorescent staining assay showed the cell viability of MGE were 81.3±2.98% and 79.5±3.56%, respectively. Ethylene glycol is a permeable cryoprotectant (PC). In the process of adding PCs, driven by osmotic pressure, the intracellular water oozes from the cells and the PCs penetrate into the cells at the same time.[9] In addition, each molecule of ethylene glycol has two hydroxyl groups which can form hydrogen bonds with the intracellular water molecules, so the increase of ethylene glycol concentration can decrease the freezing point of the cryopreservation system, inhibit intracellular ice crystal formation and prevent the membrane rupture.[38]Therefore, the thawed cell viability

22


Page 22 of 45

Page 23 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

increased when the ethylene glycol was added and the other experiment conditions were the same, such as MLG group and MLGE group. Moreover, according to Figure 5, when the trehalose was added and the other experiment conditions were the same, the cell viability was increased too. For example, the results of trypan blue exclusion assay showed the cell viability increased from 77.8±4.26% of MG group to 83.9±3.64% of MGT group and the results of fluorescent staining assay showed the cell viability increased from 75.6±4.02% of MG group to 82.4±2.51% MGT group. Trehalose is an impermeable permeable cryoprotectant(IPC) and the IPCs are unable to enter the cell membrane but they can form a layer of protective membrane on the surface of the cell membrane. At present, the protection mechanism of the trehalose is major based on several hypotheses [41-44], such as the “water substitution hypothesis”, the “preferential exclusion hypothesis” and the “vitrification hypothesis”. According to the “water substitution hypothesis”, when the cells dehydrate, the trehalose can form hydrogen bonds with the biological macromolecules from intracellular fluid and extracellular fluid, and then the trehalose membrane is formed on the cell surface and protects the cells in freezing process. According to the “preferential exclusion hypothesis”, the trehalose is difficult to form hydrogen bonds with biological macromolecules. But it forms hydrogen bonds with water molecules, and then the structure of trehalose membrane on the cell surface becomes tighter in the cooling process and the trehalose membrane can resist the adverse effects of the external environment. The results of the thawed RSC96 cells viability which were cryopreserved in supramolecular gel system showed the addition 23


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of trehalose also could reduce the ice injury, the osmotic injury and the injury caused by the recrystallization of ice crystals. MTT Colorimetric Assay In this paper, MTT colorimetric assay was used to detect the viability and growth of thawed RSC96 cells. And figure 6 showed the viability of cells increased with time from 0.5 day to 5 days.

Figure 6. The MTT colorimetric assay of the thawed RSC96 cells. A: 10 vol% DMSO was used in the experiment groups. B: 6 vol% DMSO was used in the experiment groups. The experimental groups (CB, MB, CG, MG, MGE, MGT, MLB, MLG, MLGE, MLGT) were shown in Table.1

24


Page 24 of 45

Page 25 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

As shown in Figure 6, compared to the normal cryopreservation, the trend of the cell viability did not change after the BDT was added when the thawed RSC96 cells were cultured from 0.5 day to 5 days. For example, the optical density (O.D.) value of CB increased from 0.0486 ± 0.0068 to 0.3969 ± 0.0233 when the thawed RSC96 cells were cultured from 0.5 day to 5 days, the O.D. value increased by about 7.17 times. The O.D. value of CG in which BDT was added and the other conditions were the same as CB increased from 0.0825 ± 0.0254 to 0.4854 ± 0.0338 when the thawed RSC96 cells were cultured from 0.5 day to 5 days, the O.D. value increased by about 4.88 times. As shown in Figure 2B, because the gelation temperature of BDTC supramolecular gel was 8.36 °C and the thawing temperature was 37 °C, the BDTC supramolecular gel could convert from gel to liquid and be removed easily during the thawing process. So, the adding of BDT gelator did not hinder the adherence, growth and proliferation of thawed cells. The results of Figure 6 showed the O.D. value of CB was 0.3968 ± 0.0233 and the O.D. value of CG was 0.4854 ± 0.0338 after cultured for 5 days. The main injury in the process of thawing is the recrystallization of ice crystals and the osmosis of the cryoprotectant. When the gelator BDT was added, the BDTC supramolecular gel membrane self-assembled by BDT would encapsulate the cells so that a protective layer was formed outside of the cells. The water molecules were immobilized in the BDTC supramolecular gel network, so the recrystallization of ice crystals was inhibited and the permeation rate of the cryoprotectant was decreased. In the process of thawing, the extracellular ice crystals began to dissolve and the cells were still 25


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 45

encapsulated by BDTC supramolecular gels, so that the cell injury caused by ice freezing and thawing could be reduced. Thus, when the concentration of DMSO was the same, the cell viability was increased after the gelator BDT was added. In addition, as shown in Figure 6, the O.D. value of CB was 0.3968 ± 0.0233 and the O.D. value of MB was 0.4062 ± 0.0267 after the thawed cells were cultured for 5 days. When the microchannel was used and the other conditions were the same, the MG (the O.D. value was 0.603 ± 0.0466) had the higher cell viability than CG (the O.D. value was 0.4854 ± 0.0338). And the MLG (the O.D. value was 0.526 ± 0.0467) which had the lower concentration of DMSO than CG (the O.D. value was 0.4854 ± 0.0338) had the higher cell viability. Because when BDT gelator self-assembled in the confined space of microchannel, the 3D-network structure of the BDTC supramolecular gel was more compacted. The recrystallization of ice crystals was greatly inhibited in the smaller pores of the BDTC supramolecular gel. So the results showed the compact microstructure of supramolecular gel could protect the cells during the thawing process and the cells could proliferate better when the microchannel was used. As shown in Figure 6, after cultured for 5 days, the O.D. value of MG was 0.603 ± 0.0466 and the O.D. value of MGE was 0.6215 ± 0.0457. When the trehalose was added and the other conditions were the same, MGT (the O.D. value was 0.6326 ± 0.0367) and MLGT (the O.D. value was 0.6153 ± 0.0413) had the higher cell viabilities than MG (the O.D. value was 0.603 ± 0.0466) and MLG (the O.D. value was 0.526 ± 0.0467), respectively. As mentioned in the references, 26


[45]

the

Page 27 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

multicomponent cryoprotectant can increase the content of bound water and reduce the formation of ice crystals. Ethylene glycol is a kind of hydrophilic substance with a low freezing point, it can reduce the freezing point of the intracellular water after it penetrates into the cells, so that the formation of ice crystals is reduced. Trehalose is a kind of non-permeable cryoprotectant, it cannot penetrate into the cells, but it can form a preservative membrane on the cells’ surface and protect the RSC96 cells during the removing of CPAs in thawing process. So in our experiments, when the DMSO and ethylene glycol or the DMSO and trehalose were added together, the cell viability was increased whether in cryogenic vials or in microchannel. CONCLUSIONS In this work, we used a novel method to conduct the cell cryopreservation in the microchannel. The confined space of microchannel could make the three-dimensional network structure of the BDTC supramolecular gels more compact so that the growth of the ice crystal was confined and the freezing point of the cryopreservation system was decreased. Compared to the traditional methods, the cell cryodamage could be minimized by controlling the supramolecular gel microstructure via microchannel. In addition, we also found that the multicomponent cryoprotectant system had better protective effect than single component cryoprotectant system. The IPCs trehalose was used in combination with PCs DMSO in the cell cryopreservation, it could protect the cells more effectively in the freezing and thawing process to maintain the cell activity and function. Thus, we expect that the novel BDTC supramolecular gel cryopreservation system in microchannel can be wildly used in the cryopreservation 27


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of cells, tissues or organs and can be applied to promote the development of cell therapy, tissue engineering and organs transplantation.

Associated Content Supporting Information Supporting Information is available from the Wiley Online Library. Author Information Corresponding Author *E-mail: [email protected]. ORCID Wanyu Chen: 0000-0002-5964-1254. Author Contributions ‡: These authors contributed equally. Notes The authors declare no competing financial interest. Acknowledgements This research was funded by the National Natural Science Foundation of Hubei, China (2016CFB465). References

[1] Murphy, S. V; Atala, A. Organ engineering--combining stem cells, biomaterials, and bioreactors to produce bioengineered organs for transplantation. Bioessays, 2013, 35, 163-172. 28


Page 28 of 45

Page 29 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

[2] Onofre, J; Baert, Y; Faes, K; Goossens, E. Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation. Human Reproduction Update, 2016, 22, 744-761. [3] Tonus, C; Connan, D; Waroux, O; Vandenhove, B;

Wayet, J; Gillet, L ;

Desmecht, D; Antoine, N ; Ectors, F. J; Grobet, L. Cryopreservation of chicken primordial germ cells by vitrification and slow-freezing: a comparative study. Theriogenology, 2016, 88, 197-206. [4] Yoon, S. J; Rahman, M. S; Kwon, W. S. Park, Y. J; Pang, M. G. Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome. Plos One, 2016, 11, (3):e0152690. [5] Weng, L. D; Tessier, S. N; Smith, K; Edd, J. F; Stott, S. L; Toner, M. Bacterial Ice Nucleation in Monodisperse D2O and H2O-in-Oil Emulsions. Langmuir, 2016, 36, 9229-9236. [6] Shi, Y; Zhang, Z. M; Yu, H; Huang, Y. X. The Development of the Cryopreservation

System

of

Biological

Materials.

Cryogenics

and

Superconductivity, 2005, 33, 51-54. [7] Imrat, P; Suthanmapinanth, P; Saikhun, K. Mahasawangkul, S; Sostaric, E; Sombutputorn, P; Jansittiwate, S; Thongtip, N; Pinyopummin, A; Colenbrander, B; Holt, W. V; Stout, T. A. E. Effect of pre-freeze semen quality, extender and cryoprotectant on the post-thaw quality of Asian elephant (Elephas maximus indicus) semen. Cryobiology, 2013, 66, 52-59. [8] Barbas, J. P; Mascarenhas, R. D. Cryopreservation of domestic animal sperm 29


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cells. Cell and Tissue Banking, 2009, 10, 49-62. [9] Cui, X; Labarrere, C; He, L; Cheng, S. X; Siderys, H; Kovacs, R; Gao, D. Y. Cryopreservation and Microsurgical Implantation of Rabbit Carotid Arteries. Cell Preservation Technology, 2002, 1, 121-128. [10] Pasquinelli, G; Foroni, L; Buzzi, M. Tazzari, P. L; Vaselli, C; Mirelli, M; Gargiulo, M. Smooth muscle cell injury after cryopreservation of human thoracic aortas. Cryobiology, 2006, 52, 309-316. [11] Lee, J; Kong, H. S; Kim, E. J. Youm, H. W; Lee, J. R; Suh, C. S; Kim, S. H.Ovarian injury during cryopreservation and transplantation in mice: a comparative study between cryoinjury and ischemic injury. Human Reproduction, 2016, 31, 1827-1837. [12] Bastiancich, C; Vanvarenberg, K; Ucakar, B. Pitorre, M; Bastiat, G; Lagarce, F; Preat, V; Danhier, F. Lauroyl-gemcitabine-loaded lipid nanocapsule hydrogel for the treatment of glioblastoma. Journal of Controlled Release, 2016, 225, 283-293. [13] Doméjean, H; Saint Pierre M. D; Funfak, A. Atrux-Tallau, N; Alessandri, K; Nassoy, P; Bibette, J; Bremond, N. Controlled production of sub-millimeter liquid core hydrogel capsules for parallelized 3D cell culture. Lab on A Chip, 2016, 17, 110-119. [14] Wang, X. H; Xu, H. R. Incorporation of DMSO and dextran-40 into a gelatin/alginate hydrogel for controlled assembled cell cryopreservation. Cryobiology, 2010, 61, 345-351. 30


Page 30 of 45

Page 31 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

[15] Kanmani, P; Kumar, R. S; Yuvaraj, N; Paari, K. A; Pattukumar, V; Arul, V. Cryopreservation and microencapsulation of a probiotic in alginate-chitosan capsules

improves

survival

in

simulated

gastrointestinal

conditions.

Biotechnology and Bioprocess Engineering, 2011, 16, 1106-1114. [16] Chen, W. Y; Shu, Z. Q; Gao, D. Y; Shen, A. Q. Sensing and Sensibility: Single‐Islet‐based Quality Control Assay of Cryopreserved Pancreatic Islets with Functionalized Hydrogel Microcapsules. Advanced Healthcare Materials, 2016, 5, 223-231. [17] Dean, S. K; Yulyana, Y; Williamsl, G; Sidhu, K. S; Tuch, B. E. Differentiation of encapsulated embryonic stem cells after transplantation. Transplantation, 2006, 82, 1175-1184. [18] Zhon, Y; Cui, H. J; Shu, C; Ling, Y; Wang, R; Li, H. M; Chen, Y. D; Lu, T; Zhong, W. Y. A supramolecular hydrogel based on carbamazepine. Chemical Communications, 2015, 51, 15294-15296. [19] Li, F; He, J. L; Zhang, M. Z; Ni, P. H. A pH-sensitive and biodegradable supramolecular

hydrogel

constructed

from

PEGylated

polyphosphoester-doxorubicin prodrug and α-cyclodextrin. Polymer Chemistry, 2015, 6, 5009-5014. [20] Nguyen, M. K; Alsberg, E. Bioactive factor delivery strategies from engineered polymer hydrogels for therapeutic medicine. Progress in Polymer Science, 2014, 39, 1235-1265. [21] Tanaka, A; Fukuoka, Y; Morimoto, Y; Honjo, T. Koda, D; Goto, M; 31


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Maruyama, T. Cancer Cell Death Induced by the Intracellular Self-Assembly of an Enzyme-Responsive Supramolecular Gelator. Journal of the American Chemical Society, 2015, 137, 770-775. [22] Zeng, J; Yin, Y. X; Zhang, L; Hu, W. H; Zhang, C. C; Chen, W. Y. A Supramolecular Gel Approach to Minimize the Neural Cell Damage during Cryopreservation Process. Macromolecular Bioscience, 2015, 16, 363-370. [23] Hu, Y. D; Wang, S. B; Abbaspourrad, A; Ardekani, A. M. Fabrication of Shape Controllable Janus Alginate/pNIPAAm Microgels via Microfluidics Technique and Off-Chip Ionic Cross-Linking. Langmuir, 2015, 6, 1885-1891. [24] Kini, G. C; Lai, J; Wong, M. S; Biswal, S. L. Microfluidic Formation of Ionically Cross-Linked Polyamine Gels. Langmuir, 2010, 9, 6650-6656. [25] Hubel, A. Advancing the preservation of cellular therapy products. Transfusion, 2011, 51, 82S-86S. [26] Begolo, S; Shen F; Ismagilov, R. F. A microfluidic device for dry sample preservation in remote settings. Lab on A Chip, 2013, 13, 4331-2342 [27] Toh, Y. C; Zhang, C; Zhang, J; Khong, Y. M; Chang, S; Samper, V. D; van Noort, D; Hutmacher, D. W; Yu, H. R. A novel 3D mammalian cell perfusion-culture system in microfluidic channels. Lab on A Chip, 2007, 7, 302-309. [28] King, K. R; Wang, S. H; Irimia, D; Jayaraman, A; Toner, M; Yarmush, M. L. A high-throughput microfluidic real-time gene expression living cell array. Lab on A Chip, 2006, 7, 77-85. 32


Page 32 of 45

Page 33 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

[29] Zhang, J; Wu, J; Lin, H. F; Chen, Q. S; Li, J. M. An in vitro liver model on microfluidic device for analysis of capecitabine metabolite using mass spectrometer as detector. Biosensors & Bioelectronics, 2015, 68, 322-328. [30]

Skommer,

J;

Wlodkowic,

D. Successes and

future

outlook

for

microfluidics-based cardiovascular drug discovery. Expert Opinion on Drug Discovery, 2015, 10, 231-244. [31] Xu, Y; Sato, K; Mawatari, K; Konno, T; Jang, K; Ishihara K; Kitamori, T. A Microfluidic Hydrogel Capable of Cell Preservation without Perfusion Culture under Cell-Based Assay Conditions. Advanced Materials, 2010, 22, 3017-3021. [32] Song, Y. S; Moon, S; Hulli, L; Hasan, S. K; Kayaalp, E; Demirci, U. Microfluidics for cryopreservation. Lab on A Chip, 2009, 9, 1874-1881. [33] Zhou, X. M; Liu, Z; Liang, X. M; Shu, Z. Q; Du, P. G; Gao, D. Y. Theoretical investigations of a novel microfluidic cooling/warming system for cell vitrification cryopreservation. International Journal of Heat & Mass Transfer, 2013, 65, 381-388. [34] Guven, S; Lindsey, J. S; Poudel I; Chinthala, S; Nickerson, M. D; Gerami-Naini, B; Gurkan, U. A; Anchan, R. M; Demirci, U. Functional Maintenance of Differentiated Embryoid Bodies in Microfluidic Systems: A Platform for Personalized Medicine. Stem Cells Translational Medicine, 2015, 4, 261-268. [35] Zou, Y. J; Yin, T. L; Chen, S. J; Yang, J; Huang, W. H. On-chip cryopreservation:

a

novel

method

for

ultra-rapid

33


cryoprotectant-free

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cryopreservation of small amounts of human spermatozoa. Plos One, 2013, 8, e61593. [36] Chen, W. Y; Yang, Y. J; Lee, C. H; Shen, A. Q. Conﬁnement Effects on the Self-Assembly of 1,3:2,4-Di-p-methylbenzylidene Sorbitol Based Organogel. Langmuir, 2008, 24, 10432-10436. [37] Ma, X. D. Synthesis and Gelation Properties of Tyrosine Derivatives. Xi`an University of Science and Technology, 2012. [38] Sommerfeld, V; Niemann, H. Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification. Cryobiology, 1999, 38, 95-105. [39] Ohboshi, S; Fujihara, N; Yoshida, T; Tomogane, H. Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts. Animal reproduction science, 1997, 48, 27-36. [40] Crowe, J. H; Hoekstra, F. A; Crowe, L. M. Anhydrobiosis. Annual Review of Physiology, 1992, 54, 579-599. [41] Levine, H. Another view of trehalose for drying and stabilizing biological materials. Bio Pharm, 1992, 5, 36-40. [42] Timasheff, S. N. The Control of Protein Stability and Association by Weak Interactions with Water: How Do Solvents Affect These Processes?. Annual Review of Biophysics & Biomolecular Structure, 1993, 22, 67-97. [43] Solapenna, M; Meyerfernandes, J. R. Stabilization against thermal inactivation promoted by sugars on enzyme structure and function: why is 34


Page 34 of 45

Page 35 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

trehalose more effective than other sugars?. Archives of Biochemistry & Biophysics, 1998, 360, 10-14. [44] Chen, W. Y; Yang, Y. J; Rinadi, C; Zhou, D; Shen, A. Q. Formation of supramolecular hydrogel microspheres via microfluidics. Lab on A Chip, 2009, 9, 2947-2951. [45] Wang, X. H; Paloheimo, K. S; Xu, H. R; Liu, C. Cryopreservation of cell/hydrogel constructs based on a new cell-assembling technique. Journal of Bioactive & Compatible Polymers, 2010, 25, 634-653.

35


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Only A novel supramolecular gel cryopreservation system in microchannel could minimize the cell injury during the cryopreservation process because the confined space of microchannel could make the three-dimensional network structure of the BDTC supramolecular gels more compact so that the growth of the ice crystal was confined and the freezing point of the cryopreservation system was decreased.

Schematic of supramolecular gel cryopreservation system in microchannel

36


Page 36 of 45

Page 37 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 1.The synthesis of gelator BDT. 87x57mm (300 x 300 DPI)


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. The process of cell cryopreservation and thawing in the microchannel. A: The process of cell cryopreservation. B: The process of cell thawing. 101x57mm (300 x 300 DPI)


Page 38 of 45

Page 39 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 3. The morphology of the BDTC supramolecular gel: (A) The supramolecular gel was self-assembled on the slide glass at 4 °C. (B) The supramolecular gel was self-assembled in the microchannel at 4 °C. (C) The supramolecular gel was self-assembled on the slide glass at -80 °C. (D) The supramolecular gel was self-assembled in the microchannel at -80 °C. 164x111mm (300 x 300 DPI)


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. The DSC thermograms. Control Group: DMSO (8 vol%) was added in RPMI 1640 medium. Experiment Group A: DMSO (8 vol%) and BDT(0.75 g/L) were added in RPMI 1640 medium. Experiment Group B: DMSO (8 vol%), BDT(0.75 g/L) and ethylene glycol (6 vol%) were added in RPMI 1640 medium. Experiment Group C: DMSO (8 vol%), BDT(0.75 g/L) and trehalose (0.05 mol/L) were added in RPMI 1640 medium. 296x209mm (300 x 300 DPI)


Page 40 of 45

Page 41 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 5. The results of the RSC96 cells viabilities. A: The thawed RSC96 cells were detected by the trypan blue staining assay. B: The thawed RSC96 cells were detected by Hoechst 33342 and PI double staining kit. The experimental groups (CB, MB, CG, MG, MGE, MGT, MLB, MLG, MLGE, MLGT) were shown in Table.1 288x201mm (300 x 300 DPI)


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. The results of the RSC96 cells viabilities. A: The thawed RSC96 cells were detected by the trypan blue staining assay. B: The thawed RSC96 cells were detected by Hoechst 33342 and PI double staining kit. The experimental groups (CB, MB, CG, MG, MGE, MGT, MLB, MLG, MLGE, MLGT) were shown in Table.1 288x201mm (300 x 300 DPI)


Page 42 of 45

Page 43 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 6. The MTT colorimetric assay of the thawed RSC96 cells. A: 10 vol% DMSO was used in the experiment groups. B: 6 vol% DMSO was used in the experiment groups. The experimental groups (CB, MB, CG, MG, MGE, MGT, MLB, MLG, MLGE, MLGT) were shown in Table.1 287x201mm (300 x 300 DPI)


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. The MTT colorimetric assay of the thawed RSC96 cells. A: 10 vol% DMSO was used in the experiment groups. B: 6 vol% DMSO was used in the experiment groups. The experimental groups (CB, MB, CG, MG, MGE, MGT, MLB, MLG, MLGE, MLGT) were shown in Table.1 287x201mm (300 x 300 DPI)


Page 44 of 45

Page 45 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Table of Contents 79x33mm (300 x 300 DPI)


Using a Novel Supramolecular Gel Cryopreservation System in

Recommend Documents