Breakthrough "Freeze-and-Print" Technology Debuts: Novel Cryo-Bioink Solves the Challenge of Preserving Adherent Cells
Release time:
2026-03-24
Recently, an innovative achievement by the research team led by Professor Gu Qi from the Institute of Zoology, Chinese Academy of Sciences, has brought a groundbreaking breakthrough to the fields of tissue engineering and regenerative medicine. The team successfully developed a novel biphasic bioink platform named CAMP (Cryopreservation for Adhesion and Maintenance Printing), which integrates the process of "cryopreservation, revival, and direct printing" of bioink loaded with adherent cells. This innovation completely eliminates the need for traditional toxic cryoprotectants, offering a new solution for clinical "ready-to-use" tissue repair.
Confronting the Challenge: The Dual Bottleneck of Cell Viability and Hydrogel Integrity
Combining cryopreservation technology with 3D bioprinting has long been an ideal approach for constructing tissue engineering products. It eliminates the complex in vitro culture steps required after revival in traditional methods, enabling a seamless transition from cell banks to operating rooms. However, this technology has long faced two core challenges: first, ice crystals formed during traditional freezing processes severely damage cell viability; second, the structural integrity of hydrogels used as printing matrices deteriorates after cryopreservation, directly compromising their printability. For adherent cells such as mesenchymal stem cells (MSCs), which are sensitive to mechanical forces, the loss of matrix anchoring during freezing leads to massive cell death, posing a significant barrier to technological translation. Traditional methods rely heavily on high concentrations of toxic cryoprotectants like DMSO to combat ice crystal formation, which in turn introduces severe cytotoxicity issues.
Innovative Strategy: Biphasic Design and Physicochemical Synergistic Protection
To address these challenges, Professor Gu Qi's team proposed a novel CAMP platform. The core of this platform lies in its sophisticated biphasic structural design: the inner phase consists of concave hyaluronic acid microcarriers coated with type I collagen (cHAMC), providing a stable environment for MSC attachment and proliferation; the outer phase is a hydrogel matrix composed of oxidized methacrylated alginate (OMA) that encapsulates the cells.
This design achieves dual protection at both physical and chemical levels. First, in a liquid nitrogen environment at -196°C, the OMA/gelatin matrix, rich in hydroxyl and carboxyl groups, effectively anchors water molecules through hydrogen bonding, reshaping the water network. This enhances the inhibition of ice recrystallization to approximately ten times that of conventional PBS buffer, achieving efficient, non-toxic cryoprotection without the addition of any DMSO.
Even more remarkable, the CAMP platform breaks the traditional notion that cells must be frozen in a contracted, suspended state. The microcarriers provide continuous mechanical support for the cells, maintaining them in an extended, adherent state throughout the freezing and revival process, significantly reducing apoptosis caused by osmotic pressure and mechanical stress. Experiments have shown that the cell survival rate after revival exceeds 80%.
"Freeze-and-Print": Translational Potential from Laboratory to Clinic
Another major advantage of the CAMP platform is its exceptional clinical applicability. The revived bioink can be directly used for extrusion-based 3D printing at low temperatures of 4–8°C. The dynamic Schiff base bonds between the microcarriers and the matrix within the bioink endow it with excellent shear-thinning properties, preventing nozzle clogging while ensuring smooth printing. After printing, rapid crosslinking is achieved with just 20 seconds of ultraviolet light exposure, ensuring long-term structural stability.
To validate its potential for practical treatment, the team loaded the CAMP bioink with the small-molecule drug Kartogenin (KGN), cryopreserved it in liquid nitrogen, revived it, and directly injected it in situ into distal femoral defects in rats. The results showed that this "freeze-and-print" bioink successfully induced complete subchondral bone regeneration within three months, with the rats' lower limb motor function recovering to baseline healthy levels.
Conclusion and Outlook
This study not only reveals the molecular mechanism by which maintaining cell adhesion status resists cryogenic stress—namely, by upregulating the phosphorylation level of focal adhesion kinase (FAK) to inhibit the pro-apoptotic factor JNK—but, more importantly, successfully establishes an engineered platform that integrates cryopreservation, adhesion maintenance, and on-demand printing. The findings have been published in the prestigious journal Advanced Materials.
The advent of the CAMP platform means that in the future, we may be able to "retrieve" pre-prepared cell-laden bioinks from cell banks as easily as taking out an ice cube, revive them, and immediately use them for 3D printing to construct tissue engineering scaffolds or even organ chips. This breakthrough technology offers a highly promising "ready-to-use" solution for the urgent repair of large tissue defects and the rapid fabrication of personalized implants in clinical settings, with the potential to completely bridge the critical gap from laboratory to operating room.
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