In-situ encapsulation of post-cryopreserved cells using alginate

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Applications and Health

In-situ encapsulation of post-cryopreserved cells using alginate polymers and zwitterionic betaine Jing Yang, Xiaojie Sui, Qingsi Li, Weiqiang Zhao, Jiamin Zhang, Yingnan Zhu, Pengguang Chen, and Lei Zhang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00249 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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ACS Biomaterials Science & Engineering

In-situ encapsulation of post-cryopreserved cells using alginate polymers and zwitterionic betaine Jing Yang a,b,c,d, ‡, Xiaojie Sui a,b,c,d, ‡ Qingsi Li a,b,c,d, Weiqiang Zhao a,b,c,d, Jiamin Zhang a,b,c,d,

a

Yingnan Zhu a,b,c,d, Pengguang Chen a,b,c,d, and Lei Zhang a,b,c,d*

Department of Biochemical Engineering, School of Chemical Engineering and

Technology, Tianjin University, Tianjin, 300350, China b

Frontier Science Center for Synthetic Biology and Key Laboratory of Systems

Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China c

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),

Tianjin University, Tianjin, 300350, China d

Qingdao Institute for Marine Technology of Tianjin University, Qingdao, 266235,

China. ‡ The first two authors contributed equally to this work. *Corresponding Author E-mail: [email protected]

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Keywords: cryopreservation, zwitterionic betaine, alginate polymers, In-situ encapsulation, regenerative medicine Abstract: Currently, the state-of-the-art cryoprotectants for cell cryopreservation have bottleneck problems (such as cytotoxicity), which place enormous logistical limitations to the development of regenerative medicine. In this work, the first alginate polymerbased approach for human chondrocyte cryopreservation is reported. Combined with zwitterionic betaine, a natural osmoprotectant to offer intracellular protection, this alginate polymer-based approach can achieve ~90% cryopreservation efficiency. Owing to the biocompatibility of alginate polymers and betaine, this approach can easily retrieve the post-thaw cells without traditional multistep cryoprotectant washing procedures, which is highly favorable to cell therapy. Meanwhile, owing to the feasible and mild gelation process of alginate polymers, this approach can also directly encapsulate the post-thaw cells into hydrogels without cryoprotectant removal, which is highly useful to tissue engineering. Moreover, these hydrogels exhibit tunable mechanical properties, and can form variable shapes and sizes of scaffolds in order to be injected into defect sites of patients. After encapsulating post-thaw cells, these hydrogels can maintain high cell viability (~90%) and normal cellular functions for at least 14 days. This work provides a step-change in cryopreservation of cells to be directly used in cell-based applications, and may realize the promising cellular therapy products which can integrate preservation with clinical practice.


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1. Introduction Cell-based regenerative medicine (including cell therapy and tissue engineering) offers a radical new approach to replace or regenerate the damaged tissues or organs.12

It can potentially ease the burden of global organ shortage, save countless lives, and

bring a huge economic opportunity.3 For example, the global market of tissue engineering can achieve over 30 billion dollars in 2018.4 Notably, to tissue engineering, cells and scaffolds are both key elements.5-6 So, the advanced cell preservation technologies, which are able to largely extend geographic cell-sharing regions, can provide an enormous logistical support for tissue engineering. Moreover, the integration of efficient cell preservation and biocompatible scaffolds have a dramatic impact on their therapeutic outcomes.3, 5 Cryopreservation is the most reliable technology for long-term cell preservation.7 However, the current state-of-the-art cryoprotectant (CPA), organic solvent dimethyl sulfoxide (DMSO), suffers from its bottleneck problem—intrinsic toxicity. It has been reported that DMSO can induce inactivity of enzyme, dysfunctions of cell metabolism, apoptosis of cells, and uncontrolled differentiation of stem cells.8-10 Therefore, after thawing, the immediate complex washing procedures for thorough DMSO removal are highly required. Unfortunately, many patients still suffer from various side effects (such as allergy and renal failure) after transplantation of the DMSO-cryopreserved stem cells, which have undergone the multiple washing steps.10-11 Meanwhile, these washing processes can also result in inevitable cell damages and loss.10 More importantly, before



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cell usage for tissue engineering, the post-washed seed cells after cryopreservation require harvest, maintenance, and proliferation on external scaffolds for some considerable time, and cellular adaption in the 3D environment provided by scaffold materials as well as cell-scaffold interactions must be considered.12-16 Therefore, it will be revolutionary for tissue engineering to explore the novel biocompatible CPAs, which can not only achieve high cryoprotection efficacy but also realize immediate cell usage after cryopreservation without CPA removal. Many efforts have been made to discover non-toxic CPAs inspired by nature, such as antifreeze proteins (AFPs), which were found in Antarctic fishes. However, AFPs provide limited benefit for cell cryopreservation. Most importantly, their immunogenicity still requires CPA removal if the post-thaw cells need to be injected into patients. At present, few significant improvements have been found to develop the biocompatible CPAs, which can achieve both high cryoprotection efficacy and direct post-thaw cell usage without the need of CPA removal. More recently, zwitterionic betaine has shown a great potential to be an alternative to DMSO.17 As a biocompatible osmoprotectant, it can promptly enter cells to provide intracellular protection, and presents strong abilities to protect cells from ice and osmotic injury during cryopreservation.17-19 But in order to achieve favorable cryopreservation efficacy, it is necessary to supplement extracellular CPAs to provide more bioprotection outside cells.20 Apart from the improved cryoprotection efficacy, a novel extracellular CPA that can directly contribute to cell-based regenerative medicine is also urgently desired.


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Recently, alginate hydrogels have been particularly attractive as scaffolds in regenerative medicine, because they can closely mimic extracellular matrix to provide a 3D environment for the support of cell survival and accommodation. Notably, several researchers have attempted to encapsulate cells into alginate hydrogels for extracellular cryoprotection, which are supplementary to toxic intracellular CPAs (e.g. DMSO) to achieve a remarkable cryoprotective effect.20-24 However, after cryopreservation, the encapsulated cells are difficult to be retrieved unless the hydrogels are dissociated. Meanwhile, the dissociation process can also result in inevitable injury to cells. Compared to the hydrogel-based cryopreservation, alginate polymers exhibit their flexibility for post-thaw cell usage. They can be easily separated by centrifugation for cell retrieval, or form cell-embedded hydrogels with a mild gelation process. Meanwhile. They exhibit a well-known biocompatibility, and can serve as a safe food additive that has been approved by the U.S. Food and Drug Administration (FDA).25-26 More importantly, due to its hydrophilic property, alginate polymers can efficiently bind water molecules, indicating its potential to depress water freezing point for cell cryoprotection. Therefore, these findings inspired us to use alginate polymers as the novel CPA for cell cryopreservation, and offered a new solution to directly encapsulate post-cryopreserved cells for tissue engineering. In this work, combined with zwitterionic betaine, alginate polymers instead of hydrogels were used to cryopreserve human chondrocytes (C28/I2), the common model cells to study tissue-engineered cartilage. Moreover, the other human somatic cell line (smooth muscle cells) was chosen to validate the versatility of novel CPAs (Figure 1). 5 ACS Paragon Plus Environment


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After cryopreservation, due to the superior biocompatibility of betaine and alginate, the multistep washing procedure could be omitted. The post-thaw cells can be directly retrieved with a simple dilution process for cell therapy. Meanwhile, they could also be encapsulated in alginate hydrogels by adding divalent cation (e.g. Ca2+) for tissue engineering.

Figure 1. Schematic drawing of cryopreservation procedures using zwitterionic betaine combined with alginate polymers, and the direct usage of post-thaw cells without CPA removal. 2. Experimental Section 2.1 Materials Betaine and sodium alginate (the molecular weight ranged from 4000 to 185000) were both purchased from Sigma-Aldrich. Calcium chloride (CaCl2) was purchased from Macklin Biochemical Technology Co., Ltd. Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), and fetal bovine serum (FBS) were all obtained from Gibco. 6 ACS Paragon Plus Environment

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Penicillin/streptomycin (P/S), trypsin-EDTA (0.025−0.01%), phosphate-buffered saline (PBS) and DMSO were all purchased from Beijing Solarbio Science and Technology Co., Ltd. Live/Dead kits were obtained from Invitrogen. Milli-Q water (18.2 MΩ cm−1) was used in all experiments. 2.2 Differential scanning calorimetry (DSC) tests DSC assessment of ice formation was conducted with 2% betaine, 4% betaine, 0.25% Alginate, 0.5% Alginate, 4% betaine + 0.25% Alginate, 4% betaine + 0.5% Alginate solutions. 5 ~ 10 mg samples were added into an aluminum pans and transferred to a DSC system (DCS 214 Polyma, NETZSCH). Then, these samples were cooled to -40°C at a rate of -10°C /min. After maintained at -40℃ for 5 min, they were heated to 10℃ at a rate of 2℃/min. Heat flows (W g-1) were measured in real time during the cooling and warming process. The water freezing points were determined at the beginning of the melting of the frozen solutions. The cryoscopic constant ( K f ) was calculated by the following equation, K f =T f / bB

(1)

where the T f was the depression of freezing point, bB was the molality of a solute. The total water content ( wtc ), freezing water content ( w f ) and non-freezing water content ( wnf ) in each sample were determined according to the following equations.2729

wtc  mw / m

(2)

w f  H / H w

(3)

wnf  wtc  w f

(4) 7 ACS Paragon Plus Environment


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Where mw and m represented the mass of water and the total weight of each sample, respectively, H was the melting enthalpy of each sample during warming process, which was calculated by integration of the endothermic peak, H w was the specific heat of water fusion equal to 334 J/g. 2.3 Cell preparation Human C28/I2 chondrocytes were cultured in DMEM supplemented with 10% (V/V) FBS and 1% P/S at 37°C under an atmosphere 5% CO2. SMCs were cultured in DMEM/F12 supplemented with 10% (V/V) FBS and 1% P/S at 37°C under an atmosphere 5% CO2. At a confluence of 90%, cells were detached from the culture flasks with trypsin-EDTA and collected by centrifugation at 1000 rpm for 4 min. Finally, the pelleted cells were resuspended in culture medium for further experimental use. 2.4 Osmotic regulation tests Attached human C28/I2 chondrocytes were exposed in hypertonic medium containing 1% NaCl, 1% betaine, and 1% NaCl + 1% betaine, respectively. After a 12-h exposure, cell morphology was observed by an inverted microscope (Nikon Eclipse TiS). Suspended human C28/I2 chondrocytes were exposed in hypertonic medium containing 1% betaine, 0.25% alginate, 1% betaine + 0.25% alginate, respectively. After a 6-h exposure, three fields were randomly selected to observe the cell morphology and measure the cell diameter by an inverted microscope. 2.5 Preparation of CPA solutions 8 ACS Paragon Plus Environment

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Different concentrations of betaine were dissolved in the cell culture medium, then they were sterilized using Millex-GP syringe filter units (0.22 µm, Millipore Millex). Alginate were treated with UV light overnight for sterilization and dissolved in the sterilized betaine solutions at the desired concentrations. All the solutions were kept at 4°C for further use. 2.6 Cell cryopreservation To cryopreserve human C28/I2 chondrocytes, 1×106 cells were added into 1.5 mL CPA solutions in the cryovials and incubated at 37°C for the desired period of time. After incubation, the cryovials were placed into a controlled rate freezing container at 4°C for 30 min, -20°C for 90 min and -80°C overnight, then transferred into liquid nitrogen (LN2) tank. To cryopreserve SMCs, 1×106 cells were added into CPA solutions and incubated at 37°C for the desired period of time and directly immersed into liquid tank. The cryopreservation protocol for these two cell types was based on a scientific optimization procedure. The controlled rate freezing procedure was suitable for C28/I2 chondrocytes and the rapid freezing protocol was suitable for SMCs. C28/I2 chondrocyte cryopreservation using alginate hydrogels was also conducted for comparison. After incubation, 15% CaCl2 solution was added into the cryovials to form alginate hydrogels. Then the hydrogels were cryopreserved with the same freezing procedure. 2.7 Cytotoxicity evaluation Human C28/I2 chondrocytes were exposed for 1 or 2 days to the culture medium containing 2% DMSO, 2% betaine, and 2% betaine+0.4% alginate, respectively. Then, 9 ACS Paragon Plus Environment


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they were reseeded on the 12-well TCPS plates at 37 °C under an 5% CO2. After 12 h, cell viability and attachment were tested. 2.8 Post-thaw cell retrieval and attachment tests The post-thaw cell suspension was added with 8 times amount of culture medium, then the diluted cell suspension was directly seeded into microplates. After culturing for 24 h, cell attachment function and morphology were observed by a microscope. 2.9 Cell proliferation tests The post-thaw cells were cultured in 6-well TCPS plates for 3 days. The cell numbers were counted daily using the microscope. 2.10 In-situ hydrogel formation to encapsulate post-thaw cells The post-thaw cell suspension containing CPAs in 3D printed pattern was directly crosslinked by the addition of 10% or 15% CaCl2 solutions, to form “TJU” shape hydrogels at 37 °C for 30 min. The hydrogel microparticles were generated by postthaw cell solutions containing CPAs directly crosslinked in 10 mL of 10% CaCl2 in a sterile glass container, using an electro-jetting device (a voltage of 10 kV, a 100 µL/min). 2.11 Water content tests Post-freezing alginate and betaine, unfrozen alginate and betaine, and unfrozen alginate were gelatinized by adding 10% or 15% of Ca2+. Then, these hydrogels were all punched into 1 cm diameter disks (2 mm thickness) and washed with culture medium for three times. Subsequently, these hydrogels were weighed after wiping off the


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superficial water as the wet mass ( mw ). Then, they were dehydrated at 60 °C in vacuo for 3 days and weighed again as the dry mass ( md ).30 Water contents of the hydrogel samples were calculated by the following equation.31 water content (%) = (mw  md ) / mw 100

(5)

2.12 Mechanical property tests The compressive modulus of the above-mentioned hydrogels in water content tests were measured using a microcomputer control mechanical tester (WDW-5, Beijing, China). Six hydrogel disks were compressed at a rate of 0.5 mm/min. The compressive modulus was calculated from 10% to 30% strain.30, 32 2.13 Cell viability assays The cell viability was evaluated using live/dead assays (Live/Dead viability kit). The cell suspension or cell-embedded hydrogels were introduced into a microplate. Then the calcein-AM/ethidium homodimer-1 reagent mixture solution was added to the microplate according to the manufacturer’s instruction. Subsequently, the microplate was incubated at room temperature for 30 min away from light, and then observed by an inverted microscope. The viability was calculated by counting the number of live cells (green) and dead cells (red). To determine cell viability in the hydrogels, the cellembedded hydrogels were gently rinsed twice with 0.9% NaCl before staining. 2.14 Long-term viability of encapsulated cells 500 μL aliquots of the post-thaw cell suspension were directly added with 500 μL 10% or 15% CaCl2 solutions to form hydrogels at 37 °C for 30 min. Then, the cell11 ACS Paragon Plus Environment


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embedded hydrogels were rinsed using the cell culture medium for three times to remove uncrosslinked Ca2+, and punched into 5 mm diameter disks and cultured with cell culture medium under an atmosphere of 5% CO2 at 37°C. 2.15 Data analysis Statistical analysis was determined using Microsoft Excel 2016. Significances were determined using a two-tailed homoscedastic Student’s t test with a 95% confidence interval. 3. Results and discussion 3.1. Ability to inhibit ice formation and enter cells During cryopreservation process, ice formation and growth are highly detrimental to cell functions, and induce final cell death.33-34 In 1972, Mazur proposed two cryoinjuries as follows: First, extracellular ice formation can lead to water efflux and increase intracellular solute concentrations, thus causing cell death due to hyperosmotic shock (known as osmotic injury). Second, intracellular ice formation can impair cellular microstructure and protein functions, thus resulting in cell death due to mechanical damages (known as ice injury).35 Therefore, novel CPAs should be capable of inhibiting ice formation for successful cryopreservation. It has been reported that water crystallization is directly associated with hydrogenbond network (HBN) formation/development by water molecules.36 So, the molecules or polymers which can break water-water hydrogen bonds should be able to inhibit ice formation/growth, and have the potential to serve as novel CPAs. Therefore, in this work, we used betaine and alginate which are both hydrophilic and capable of strong 12 ACS Paragon Plus Environment

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binding water molecules. We firstly investigated their influences on ice formation using DSC, and the results were shown in Figure 2. According to the thermograms, we evaluated their ability to depress water freezing point, and calculated the freezing water contents of their solution systems. The exothermic peaks in Figure 2a presented ice melting of alginate, betaine, and betaine-alginate solutions compared with pure water, and the smallest peak temperatures of betaine-alginate samples indicated their strongest ability to decrease water chemical potential. Figure 2b showed that betaine could efficiently depress water freezing point with a cryoscopic constant of 14.9 ℃·Kg/mol, consistent with our previous studies.17,

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Because zwitterionic betaine possessed

cationic and anionic groups, which enabled strong binding with water molecules via electrostatically induced hydration effects.38-39 Meanwhile, alginate also showed an ability of freezing point depression, resulting from the multiple hydroxyl groups in polyguluronate segments of alginate polymers. These hydroxyl groups could impede water molecule motion, prevent their accumulation for crystallization, and transform them into a non-freezing state.40 It has been reported that each unit of alginate could attach surrounding eight molecules of water, which were restrained into a non-freezing state.41 More importantly, betaine-alginate samples performed best amongst others, showing that the combination could improve the depression of water freezing point. It was consistent with Figure 2c, which presented their reduction of freezing water contents. These above data clearly showed that the combination of betaine and alginate could efficiently inhibit ice formation/growth, which was an important characteristic of the novel CPAs to prevent cryoinjuries. 13 ACS Paragon Plus Environment


Moreover, the ability of a novel CPA to enter cells could ensure intracellular cryoprotection, which is key to efficient cryopreservation. As a well-known osmoprotectant, betaine has been reported to enable enter cells by osmotic driven via transport proteins, which was confirmed by Figure 3. Under hypertonic NaCl medium (110% higher than physiological osmotic pressure), the debris of dead cells could be observed due to osmotic shock. In contrast, the addition of betaine could maintain the normal cellular morphology and growth. Because betaine could enter cells to regulate osmotic pressure and offer intracellular protection. Next, we further investigated the ability of betaine and alginate to balance intracellular and extracellular osmotic pressure. As shown in Figure S1, when suspended cells were exposed in 0.25% alginate medium for 6h, the cell diameter was significantly reduced. It was because that alginate polymers increased the extracellular osmotic pressure, causing cell dehydration. Interestingly, when adding betaine into the alginate medium for 6h incubation, the cell diameter was similar with that of fresh cells, indicating that the cell volume was maintained. It was attributed to betaine uptake by cells to balance intra/extracellular osmotic pressure.

Figure 2. DSC tests. (a) Differential scanning calorimetry (DSC) thermograms, (b) the depression of water freezing point and (c) the freezing water contents of pure water, 14 ACS Paragon Plus Environment

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0.25% alginate, 0.5% alginate, 2% betaine, 4% betaine, 4% betaine+0.25% alginate, 4% betaine+0.5% alginate solutions. B: betaine, Alg: alginate.

Figure 3. Cell morphology after 12-h exposure to the medium containing 1% NaCl, 1% betaine, 1% betaine + 1% NaCl, and pure medium as control. The red arrows indicated the debris of dead cells. B: betaine, Alg: alginate. Scale bar = 100 μm. 3.2. Cryopreservation efficacy As is well known, the self-repair ability of articular cartilage is very limited, due to its avascular and alymphatic nature.5, 42 Currently, the treatment of articular cartilage defects is still a huge challenge. Cartilage tissue engineering technology can transplant autologous/heterologous chondrocytes for regeneration of articular defects, which has been a promissing treatment method.42-43 It is commonly based on the chondrocyteseeded scaffolds to repair or replace cartilage tissues. Therefore, due to the essential role of chondrocytes in tissue engineering, maintained their viability and functions are highly required. In this work, we aimed to develop a novel technology, which was not only capable of effective chondrocyte preservation, but also capable of direct usage of chondrocyte encapsulated in hydrogel scaffords for tissue engineering. Firstly, we cryopreserved human C28/I2 chondrocytes using betaine and alginate as CPAs (Figure 4). Figure 4b showed that the optimal cryoprotection efficacy using betaine alone was 52% (post15 ACS Paragon Plus Environment


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thaw cell viability), indicating its considerable efficiency to protect chondrocytes from cryoinjuries. Moreover, after incubation and supplementation with alginate, the efficiency was further improved as shown in Figure 4c. The optimal post-thaw cell survival efficiency of betaine and alginate could achieve ~91%, demonstrating their excellent cryoprotection capability (Figure 4d). According to our earlier study, zwitterionic betaine could promptly enter cells via the protein transporters to offer intracellular cryoprotection.17 Meanwhile, supplementary alginate polymers used in this work could increase extracellular osmotic pressure to enhance betaine entry, as well as offering extracellular cryoprotection. Therefore, their combination could ensure the improved efficiency. However, when cells were cryopreserved using alginate hydrogels, the post-thaw survival efficiency (~46%) was significantly lower than that of alginate polymers (Figure S2). It was probably because that the polymer network based-hydrogels restricted the diffusion of betaine and hindered its uptake by cells.44 In contrast, alginate alone (1%) resulted in just 14.3% of cell survival after thawing, similar with the blank control sample. It suggested that during the freezing process alginate polymers with large molecule weights were unable to enter cells to offer intracellular protection. Moreover, to further validate the versatility of this CPA composition, smooth muscle cells (SMCs) were cryopreserved and also exhibited a high performance (~74% viability), as shown in Figure 5.


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Figure 4. Human chondrocyte cryopreservation. (a) Fluorescence images of the live/dead assay of C28/I2 cells. Post-thaw cell viability of chondrocytes (b) using betaine at different concentrations, (c) incubated for different time before cryopreservation with 4% betaine (blue), 4% betaine+0.75% alginate (red), 4% betaine +1% alginate (green), 4% betaine +1.25% alginate (purple), (d) incubated for 2h before cryopreservation with 4% betaine and different concentrations of alginate. B: betaine; Alg: alginate. Scale bar = 100 μm. Value =mean ± standard deviation, n≥3. *** p  0.001 .



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Figure 5. SMC cryopreservation. (a) Fluorescence images of the live/dead assay of SMCs. Post-thaw cell viability of SMCs (b) using betaine at different concentrations, (c) incubated for different time before cryopreservation with 6% betaine (blue), 6% betaine+0.75% alginate (red), 6% betaine +1% alginate (green), 6% betaine +1.25% alginate (purple), (d) incubated for 2h before cryopreservation with 6% betaine and varied concentrations alginate. B: betaine; Alg: alginate. Scale bar = 100 μm. Value =mean ± standard deviation, n≥3. *** p  0.001 , * p  0.05 . 3.3. Cytotoxicity evaluation After cryopreservation, the recovered therapeutic cells should be used for patients. Therefore, the biocompatibility of CPAs is highly required to ensure the therapeutic safety.10, 27 However, the intrinsic toxicity of the state-of-the-art CPA (DMSO) still hinders its clinical applications for cell-based regenerative medicine. In this work, we used betaine and alginate as intracellular and extracellular CPAs, which were both highly biocompatible. Zwitterionic betaine is widely distributed in plants, animals, and even human beings, while alginate is a type of natural polymer that is mainly used as a food additive approved by FDA.25-26, 45 To verify their nontoxicity, we exposed chondrocytes in culture medium containing betaine, betaine-alginate, and DMSO. After a 2-day exposure, betaine with and without alginate samples showed similar cell viability with that of blank control sample, indicating that these novel CPAs showed no observable cytotoxicity (Figure 6a). In contrast, cell viability of DMSO sample was significantly decreased, suggesting that it was harmful to cells, consistent with the numerous previous reports.27, 46 Moreover, the cells in betaine with/without 18 ACS Paragon Plus Environment

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alginate samples presented normal spindle-like shape as well as fresh ones, as shown in Figure 6b. While those in DMSO samples presented abnormal morphology with many blebs inside, probably indicating that DMSO induced cell apoptosis.

Figure 6. Cytotoxicity evaluation. (a) Cell viability and (b) attachment images of chondrocyte after exposure in culture medium containing 2% DMSO (blue), 2% betaine (red), 2% betaine+0.4% alginate (green) for 1 day (upper row), 2 days (lower row), and culture medium (control). Scale bar =250 μm. Value =mean ± standard deviation, n≥3. ** p  0.01 . 3.4. Post-thaw cell retrieval without multistep washing processes Currently, before the usage of post-thaw therapeutic cells, multiple washing steps to thorough DMSO removal are highly required.47-48 However, these washing processes not only present the complexity and time consumption, but also can lead to inevitable cell damages and loss.10 In this work, based on the highly biocompatible betaine and alginate, we proposed to skip the complex washing steps for successful cell retrieval, which would be significantly meaningful to cryopreservation applied in cell-based regenerative medicine. We directly retrieved post-thaw chondrocytes with CPA solution, which was just diluted by medium due to the limited tolerance of active cells 19 ACS Paragon Plus Environment


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to hyperosmotic exposure. Similar with fresh chondrocytes, the retrieved cells exhibited the maintained cellular morphology and proliferation (Figure 7a and 7b), suggesting that their normal functions were not affected after cryopreservation without washing process. These results showed the potential of betaine-alginate CPAs to be used for therapeutic cell products.

Figure 7. Cell retrieval without multistep washing process. (a) Attachment and (b) proliferation function of fresh and post-thaw chondrocytes without washing steps. Scale bar = 250 μm. Value =mean ± standard deviation, n≥3. 3.5. In-situ encapsulation of post-thaw cells Direct cell usage after preservation is highly favorable to cell-based therapeutic products, which will also be a revolution to tissue engineering. So, in this work, postthaw cells with CPA solutions (0.75% alginate and 4% betaine) were directly encapsulated to form different sizes and shapes of hydrogels, indicating their potential to be injected into different defect sites of patients (Figure 8a and 8b). Moreover, these hydrogels that were ionically crosslinked by post-freezing alginate could maintain their high water content (93%) and mechanical properties, similar with those crosslinked by unfrozen alginate with/without betaine, as shown in Figure 8c and 8d. Alginate 20 ACS Paragon Plus Environment

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hydrogels were reported to possess tissue-like elasticity to mimic extracellular matrix, having been widely used as scaffolds in cartilage tissue engineering. Therefore, this result suggested that the functions and properties of alginate polymers as CPAs were not affected after the freeze-thaw cycle, and they could form good-performance alginate hydrogels same with the control tissue-like hydrogels. Meanwhile, the addition of betaine could not influence the water contents, because the smaller betaine molecule would diffuse out of the polymer network-structured hydrogels. Furthermore, the postfreezing alginate-based hydrogels could maintain the similar characteristics with unfrozen control samples at the different crosslinking densities (10% or 15% Ca2+).

Figure 8. In-situ hydrogel formation to encapsulate post-thaw chondrocytes. (a), (b) Photo and fluorescence images of different sizes and shapes of cell-embedded alginate hydrogels after cryopreservation. (c) Water content and (d) compression modulus of 21 ACS Paragon Plus Environment


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hydrogels ionically crosslinked by unfrozen 0.75% alginate (blue), unfrozen 4% betaine+0.75% alginate (red), and frozen 4% betaine+0.75% alginate (green). Value =mean ± standard deviation, n≥3. 3.6. Long-term viability of the encapsulated cells in hydrogels After therapeutic cells encapsulated in alginate hydrogels as scaffolds, their longterm viability and functions should be maintained for tissue engineering.14 As shown in Figure 9a and 9b, the encapsulated cells showed uniform distribution in bulk hydrogels (presented in Figure 9c) from Day 0 to Day 14. At the time of hydrogel formation using 4% betaine and 0.75% alginate, ~80% of the cells were alive (cryopreservation efficacy was 81.7%), suggesting that the gelation process was highly mild. After 4-day culture, cell viability was increased to ~90%, indicating that the encapsulated cells gradually adopted the 3D environment of post-freezing alginate-based hydrogels. These hydrogels comprised the biocompatible polymer networks, which could provide a highly swollen environment similar to native tissues. Meanwhile, they could also allow the free diffusion of nutrients and metabolic waste to support and accommodate cell survival. Figure 9d showed that the cells could maintain their viability around 90% for at least 14 days. After 14-day culture, the cells were retrieved by dissolving hydrogels, and reseeded in 6-well TCPS plates. As shown in Figure 9e, the retrieved cells were found to attach onto the substrate, and could maintain their membrane integrity and cellular morphology similar with fresh cells. Moreover, they could proliferate normally as well as fresh cells (Figure 9f). These results indicated that after cryopreservation,


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encapsulation, and long-term culture in hydrogels, the functions of cells including attachment and proliferation were not affected.

Figure 9. Long-term viability of encapsulated cells in hydrogels. (a) Phase contrast images and (b) fluorescent images with live/dead assay of hydrogel-encapsulated cell. (c) Photo images of the in-situ forming bulk hydrogels to encapsulate post-thaw cells 23 ACS Paragon Plus Environment


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with CPAs. (d) The viability of encapsulated cells in alginate hydrogels at different crosslinking densities for certain time. (e) Cell attachment and (f) proliferation of the retrieved chondrocytes. Value= mean ± standard deviation, n≥3. Scale bar = 100 μm. Value =mean ± standard deviation, n≥3. 3.7. Proposed mechanism Based on the above results, a potential mechanism could be proposed. When chondrocytes were cryopreserved using alginate polymers alone, it could offer a minor extracellular cryoprotection, because its hydrophilic property could inhibit ice formation. Commonly, sucrose and trehalose with a molecular weight of 343 were reported to serve as extracellular CPAs, due to their inability to penetrate cell membrane.49-50 Obviously, alginate polymers with much bigger molecular weight was unable to permeate cellular phospholipid bilayer. Meanwhile, no specific transporters for alginate were reported on mammalian cell membrane. Therefore, alginate could not provide intracellular cryoprotection, which was the key point of successful cryopreservation, finally leading to cell death (Figure 10a). When chondrocytes were cryopreserved using betaine alone, it could offer the considerable intracellular and extracellular protection to chondrocytes from cryoinjuries. Betaine was able to prevent ice formation. Moreover, it can enter cells under a hypertonic stress due to its osmotic regulation ability.17-18 Therefore, during the freezing process, betaine inside and outside cells could provide considerable


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intracellular and extracellular cryoprotection to enable a higher post-thaw cell survival rate.(Figure 10b). When chondrocytes were cryopreserved using betaine combined with alginate, they could achieve excellent intracellular and extracellular protection to chondrocytes from cryoinjuries. Alginate polymers in medium could increase extracellular osmotic pressure, which promoted betaine uptake by cells and enhanced intracellular protection. Moreover, the combination of betaine and alginate could strengthen the ability to inhibit ice formation. Therefore, during freezing-thaw cycle, enhanced intracellular and extracellular protection could be achieved, resulting in the extraordinary cryoprotection efficiency. After cryopreservation, extracellular alginate could be directly ionically crosslinked to encapsulate post-thaw cells into hydrogels without the need of CPA removal, and form the mimic of extracellular matrix to support cell survival for a long time. During the nutrient/waste exchange through hydrogels in long-term incubation, intracellular CPA betaine would diffuse out of the polymer networks (Figure 10c).



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Figure 10. Proposed mechanism. 4. Conclusions In this work, the first alginate polymer-based approach for effective cell cryopreservation was reported. By combining the zwitterionic betaine, the post-thaw cell viability could achieve ~90%. Because alginate polymers could provide extracellular protection and betaine could enter cells via membrane transport proteins to offer intracellular protection. Moreover, due to the unique property of alginate polymers, the post-cryopreserved cells could be directly used in cell-based regenerative medicine.


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Other alginate hydrogel-based cryopreservation approaches have been reported, but they face the challenge of the utilization of toxic cryoprotectant and the retrieval of post-thaw cells due to the harmful and time-consuming hydrogel dissociation.20-24 To solve this problem, we first used biocompatible alginate polymers as extracellular cryoprotectants, which were combined with zwitterionic betaine instead of toxic organic solvents as intracellular cryoprotectants. Due to the biocompatibility and mild gelation process of alginate polymers, the multistep washing procedure for cryoprotectants removal could be omitted. The post-thaw cells could be easily retrieved with a simple dilution method or directly encapsulated into alginate hydrogels by adding divalent cation (e.g. Ca2+). It meant that this alginate polymer-based approach could integrate the efficient cell cryopreservation with direct usage of therapeutic cells, which was highly meaningful to provide the powerful support to develop popular therapeutic products to benefit the patients in hospitals, clinics, or even home in the near future. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge. Figure S1: The effect of betaine with/without alginate on osmotic pressure. (PDF) Figure S2: Live/dead assay of post-thaw C28/I2 cells cryopreserved with alginate polymers and alginate hydrogels. (PDF)



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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡Jing Yang and Xiaojie Sui contributed equally. Notes The authors declare no conflicts of interest. ACKNOWLEDGMENT The authors acknowledge the financial support from the National Natural Science Funds for Innovation Research Groups (21621004), National Natural Science Funds for Excellent Young Scholars (21422605), the Qingdao National Laboratory for Marine Science and Technology (QNLM2016ORP0407), Tianjin Natural Science Foundation (18JCYBJC29500). REFERENCES 1.

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In-situ encapsulation of post-cryopreserved cells using alginate polymers and zwitterionic betaine Jing Yanga,b,c,d, ‡,Xiaojie Suia,b,c,d, ‡Qingsi Li a,b,c,d, Weiqiang Zhao a,b,c,d, Jiamin Zhanga,b,c,d,Yingnan Zhua,b,c,d, Pengguang Chen a,b,c,d, and Lei Zhanga,b,c,d* TOC/Abstract Graphics


In-situ encapsulation of post-cryopreserved cells using alginate

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