Introduction
The potential of 3D bioprinting does not depend only on its applied ability to adopt the form and anatomy of biological tissues, but in regard to creating real transplantable substitutes of human organs. The pathway towards clinically accepted bioengineered tissues is however complicated, beginning with proof-of-concept prototypes. Cell viability is one of the numerous technical and biological challenges along this trail that has turned out to become one of the most critical determinants that can make bioprinted constructs either succeed or fail in the end in the realm of therapeutics.
The security institutes located all over the world are optimizing bioinks, printer parameters and fabrication approaches of this technology in order to reduce cell stress and enhance long-term functioning. However, when it comes time to scale up constructs and be ready to run clinical trials, the focus is more on how the printed cells can not only survive the process of printing, but also the challenges of integration, immunocompatibility and the natural challenges of functional regeneration of the cell within the human body.
This article discusses the relevance of high cell viability venturing across bioprinting pipeline. It relates technical requirements with clinical end-user needs, the biological and regulatory issues of the lab to the bedside translation of tissues cultured in the laboratory, and the emerging research that is influencing tomorrow of regenerative medicine.
What is Cell Viability and Why Does it Matter?
Viability of cells is the ratio of the number of live healthy cells in a specific population at a certain time. In reference to bioprinting practice, it refers to the degree of successful survival of the cells during the preparation, extrusion/ deposition process and then the maturation within a scaffold or a tissue matrix.
The bio-printed cells should not only endure the mechanical or thermal pressures of the printing procedure; additionally, they:
- Replicate so as to regenerate tissue structures
- Differentiate into functional cells specialised into cells
- Signal through signals pathways
- Support metabolic drives and carry homeostatic functions
These are hampered by low cell viability. Stressed cells, apoptotic cells or necrotic cells will lead to tissue degradation, inflammation or a total construct failure. In multi-layered tissue models, especially vascularized liver lobules, cortical brain organoids, or cardiac patches, when viable cell population in the core is lost, due to lack of oxygen and ultimately rejection on implantation, there is impaired functionality.
Conversely, high cell viability raises the success rate of engraftment and facilitates the long-term incorporation with host tissue and augments the therapeutic potentials of the printed construct. It renders it an inviolable variable in the medical preparedness of bioprinted medication.
Technical Contributors to Cell Viability
Biopriming Modality
The various methods of bioprinting appreciate dissimilar levels of mechanical or thermal stress on cells.
- Extrusion Bioprinting: Applies pressure to have cell loaded bioink injected through a nozzle. Here shear stress may cause damage to the cell membrane or may distort cytoskeleton organization.
- Inkjet Bioprinting: Speeds up the drops through the means of thermal or piezoelectric energy. The high temperatures to which the cells are exposed risk being exposed due to the thermal variants.
- Laser Assisted Bioprinting: The nozzle-free method of laser pulses deposition. This tends to avoid a low cell viability as long as there is a precise calibration of the laser energy.
Both methods need to be considered in the light of their effect on cell survival and behaviour of various cell types. Stem cells, in particular, respond better to shear changes in addition to thermal changes as opposed to the fibroblast or the endothelial cells.
Bioink Composition
Bioinks have a duple purpose of acting as both a literal scaffold and a biological microenvironment of cells. Both the viscosity and cross-linked behaviour as well as degradation -rate affects the responses of the cells during and after printing. Over-viscous bioinks need greater extrusion pressure that induces greater mechanical stress. Conversely, low viscosity inks can have no structural stability and will undermine cell anchorage and orientation.
The applicability of biocompatible hydrogel (e.g. gelatin-methacrylate (GelMA), alginate, fibrinogen) has extensive application. The latest innovation in developing bioinks is the development of cell-responsive formulation in which cell survival and maintenance of phenotype are encouraged.
Environmental Conditions
Post-printing cell recovery is directly influenced by temperature, the concentration of oxygen, pH, nutrient availability in the printer condition, and culture condition. Given a soft printing process, even a given poor incubation, or less-than-optimal perfusion, may result in loss of viability given time. To overcome this bioreactors and perfusion systems were introduced capable of providing continuous media flow and mechanical stimulation, both of which have been shown to help maintain high viability as tissues undergo a maturation process.
Scaling the Challenge: From Small Constructs to Clinical Tissues
Although the stand-alone cell survival in small-scale experimental constructs is a complicated task, the task becomes exponentially more complicated as the experimental setup grows to the tissue size suitable to be used by humans. The bigger tissues encounter issues such as:
- Diffusion Limitation: Without active perfusion the movement of oxygen and nutrients within the hydrogel is restricted to 100-200 mu m. Cells in the middle of thick structures are at risk of dying out before vascularization will occur.
- Heterogeneity of Cell Types: Various cells can have various requirements of different growth factors, degree of matrix stiffness, or level of oxygen. Running these across a construct are more prone to imbalance large-scale.
- Prolonged Culture Times: Developing bigger tissues may persist to take weeks or months. There is also a need to sustain high viability during this time through optimizing media, mechanical support and bioactive signalling.
Clinical-grade products as well need to conform to the standards of GMP (Good Manufacturing Practice), and that requires reproducibility, sterility and production of strictly controlled variability between batches, all of which is influenced by the quality of cell survival and functional properties during a production run.
Clinical Implication of Cell Viability
Engraftment and Integration
When a tissue produced by means of bioprinting is implanted into the body of a human being, this tissue can directly depend on the quality of the cell population structure in the corresponding area of survival after transplantation. Poorly viable tissues would either be rejected, provoke an inflammatory reaction, or simply do not merge with the structures around. The viable cells on the other hand release cytokines and extracellular matrix products that encourage interaction with the host, angiogenesis and stabilization of functions.
Immune Response
Death or dying cells cause the release DAMPs, which activates the immune system, or damage-associated molecular patterns. This inflammatory response may go up to graft rejection or whole-body complications. Some of these adverse signals can be avoided by maintaining cell viability to enhance compatibility particularly in allogeneic or xenogeneic constructs.
Long Term Functionality
But survival is not enough; clinical efficacy also needs bioprinted tissues to conduct organ-specific functions: a cardiac patch must contract, liver tissue must metabolize drugs and pancreatic tissue must secrete insulin. The functions argue not only with presence of cells, but also their health, age and communication with other type of cells. The ability to survive (the high viability) increases the likelihood that the cells would continue to perform their respective expert functions.
Regulatory Considerations
With bioprinted tissues moving closer to clinical trials the FDA, EMA, and the WHO regulatory agencies are putting cell viability measures under the microscope. Although there is as yet no standard measure of minimum viability, there is the indication sought by regulators of:
- Verified viability assays (such as; MTT, Trypan blue exclusion, ATP luminescence)
- Illustrated living ability of living cells (e.g., metabolic activities, gene [protein expression)
- Consistency in batch production among various productions
- Information displaying viability perseverance over clinical relevant timeframes
Also, an increasing priority is placed on the process of standardizing definitions, measures, and reporting of viability. Agencies are coming into demand evidence of cell survival being in accordance with intended profile and safety before the use is given a pass in the form of trial.
Current Research Trends and Innovations
A number of new technologies are directed to enhance cell viability in bioprinting:
- Microfluidic Bioprinting: Combines the control of fluid and organization of space in order to reduce shear and better distribute nutrients.
- Oxygen Releasing Bioinks: Provision of oxygen to cells temporarily when there is absent vasculature at the initial stages of post-printing.
- 4D Bioprinting: Adapts stimuli-affected materials which alter in shape or action, in turn, following implantation, improving flexibility and survival.
- Real Time Viability Monitoring: Places biosensors into constructs and evaluates conditions of viability and corrects them dynamically in the course of developing tissue.
Studies are also underway into hybrid bioprinting platforms, which simultaneously integrate two or more modalities, e.g. laser-assisted and extrusion printing, to exploit both high-resolution placement and bulk tissue scalability, along with minimal viability losses.
The Future of Viable Bioprinted Therapies
Bioprinting is quickly developing in the health care environment. Bioprinted skin, cartilage and vascular graft pilot-studies are well underway. Although the majority of the developed applications are still experimental or at their initial stages of clinical trials, the reports of success are starting to come in.
As an example, airway splints which are made by 3D bioprinting and tailored to the needs of a patient have demonstrated positive effects on children cases, mostly because of the long-lived stability of living parts used. In parallel, bioprinted patch-based solutions to repair of the myocardium are being tested to promote contractility after infarction, with viability parameters as the key determinants in trial eligibility.
In possible ways in the future, high-viability printing can produce organoids that can be utilized in drug testing to personalize treatment approaches in cancer or genetic illness with reduced reliance on animal models and the attendant dissimilarities of animal models to people.
Conclusion
The shift between the lab-based tissue prototypes toward life-saving clinical implants rests on a single principle: the preservation of cells alive, active and responding by the end of the bioprinting procedure. Cell viability is in itself a predictor of long-term integration, safety and therapeutic success instead of a mere measure of short-term survival.
Be it a better scaffold design, better bioinks, or better prints, any step taken in bioprinting will have to be squarely in the biological requirements of the cells as its central focus. Integrating the viability into the core of biofabrication, it will help researchers and clinicians to bridge the gap between the promise and the practice, thus affecting regenerative solutions out of the bench and into the bedside.
