Cells or tissues product can be preserved for extended periods through cryopreservation [23]. Successful cell cryopreservation relies on proper freezing, sufficient storage, and accurate thawing methods [24]. Cryopreservation can lead to various types of cell damage through different mechanisms. Ice crystal formation, growth, and recrystallization are major limitations in cryopreserving cells, tissues, and organs, leading to severe damage to the biological samples [25]. Osmotic shock occurs as ice formation draws water out of cells, leading to cellular dehydration, shrinkage, and membrane damage [26]. Oxidative stress during cryopreservation also further damages cellular components. After thawing, cells must recover by controlling thawing to prevent ice recrystallization, gradually rehydrating to avoid osmotic shock, and activating repair mechanisms like DNA repair and antioxidant defenses [14]. Effective cryopreservation involves balancing these factors to minimize damage and aid recovery. Cryoprotectants like DMSO are commonly used to prevent ice formation, but high concentrations can be toxic, causing protein denaturation and metabolic disruption. According to other studies, the optimal DMSO concentration for cells, such as immortalized cell lines and hematopoietic stem cells, is typically between 5 and 10% [3, 27]. The usual cryo medium used in cell cryopreservation is a combination of either FBS, HPL or medium with the DMSO [28, 29].

In this study, the cell performances and viability in different cryopreservation conditions like different cryo mediums, storage durations, storage locations and cell revival methods were evaluated. Cell viability is defined as the existence of structural, metabolic and, for proliferating cells, reproductive and integrities essential for the preservation of life [24]. There are various methods to assess cell viability ranging from quantitative and qualitative measures, such as cell attachment, rate of cell growth, enzymatic activity and dye exclusion. Previous study showed that human primary skin cells can be stored via cryopreservation for up to 12 months and still retain their characteristics [1]. Fibroblast shows the highest number of vials with optimal cell attachment of more than 60% compared to other cell types as shown in Fig. 1A. Different cells respond and adapt differently to cryopreservation depending on their physical and biological properties, their origin donor as well as the metabolic condition and cell passages in an expanded primary cell culture [30]. Fibroblasts, known for their versatility and functional nature, have high viability due to their robustness, shorter doubling time, and ability to survive in vitro culture [31, 32]. On contrast, keratinocytes which are fully differentiated cells from the epidermis, grow well when cultured with fibroblasts with the synergistic effects of growth factors [33, 34]. The growth of keratinocytes benefits from being cultured with fibroblasts without using animal components and serum free medium [35, 36].

The attachment of revived cells that cryopreserved in FBS + 10% DMSO showed a higher attachment rate, optimal total number of live cells, and better viability compared to those cryopreserved in commercial medium (Figs. 1B and 2). The morphological images of fibroblasts in Figs. 3 and 4 also show that fibroblasts in the FBS + 10% DMSO group exhibited better cell attachment on day 1 in both revival methods compared to other groups. FBS provides essential nutrients, hormones, growth factors, amino acids, proteins, and other factors necessary for cell metabolism and proliferation, helping maintain cell viability during cryopreservation [37]. This is crucial for cells to survive the stress of freezing and thawing. Previous findings by Fugisawa et al. (2019) also reported that FBS helps balance osmotic pressure during freezing and reduces ice crystal damage [38]. FBS is one of the earliest and most commonly used agents in cryopreservation, especially in fields involving mesenchymal cells [39]. According to Duarte Rojas JM et al. (2024), cryopreservation and thawing of cells is more effective in either platelet lysate serum or FBS [37]. Nonetheless, the advancement in cryopreservation have exposed established risks associated with the use of calf-harvested serum such as xenogenic materials, transmission of animal to human infectious disease and immunizing effect that compromise the cells’ quality and biosafety in clinical application and therapeutic outcome [36, 40].

An alternative of serum-free media or synthetic mediums, and human-derived serums, plasma or platelet derivatives become safer preferences with impressive cell viability, sterility and extensive expansion with stable immune phenotype, differentiative and immunomodulatory, which is ideal for clinical applications [40]. The synthetic mediums are chemically defined, offering consistency, and can be tailored to different cell types improving their survival and functionality [41]. In this study, cells cryopreserved in a commercial cryo medium showed lower viability might be due to the defective CoolCell freezing container (Mr. Cool) used, which couldn’t close properly, leading to suboptimal cooling and stress to the cells. Additionally, a study reported that the percentage of cell recovery slightly dropped after being stored in commercially available medium for 5 months of cryostorage compared to 1 month, with a slight increase in caspase 3 activity, an indicator of apoptotic cells [42]. Therefore, the suitability of the cryomedia, as well as the standardization of the storage and handling processes, is crucial to ensure consistent cooling rates and minimise thermal stress, thereby preserving cell viability and functionality post-thaw.

Prolonged cryopreservation duration can affect the long-term viability [1] and genetic stability of cells. It is known that low temperatures stop the metabolic activity of cells, and cryoprotective agents are used to protect cells from damage caused by freezing temperatures. However, the longer the storage duration, the more cells are exposed to temperature fluctuations, the effectiveness of the cryo medium, and potential issues from the storage conditions themselves [7]. In this study, majority of the cells able to reach more than 60% cell attachment in 24 h for the 0–6 months storage durations whilst cells stored for more than 24 months have the most numbers of vials with less than 20% cell attachment (Fig. 1C). For short-term cryopreservation studies, the viability of HDF post-cryopreservation at 1 and 3 months showed a slight decrease trend compared to before cryopreservation, but the cells still actively proliferated within 7 days of culture (Fig. 2). This might be due to disruptions in the ultra-low temperature environment, such as issues with cryoprotective agents or external factors like frequent opening of nitrogen tanks, can cause cryoinjury to cells. This injury leads to osmotic imbalances, crystal formation, and damage to cell membranes and organelles, which are crucial for cell viability [7].

The method used to revive cells after cryopreservation also could affect cells viability. The main difference between the direct and indirect methods is the concentration of the cells obtained. The indirect method uses centrifugation to concentrate cells pellet, increasing viable cell yielded, removing the DMSO and reducing the contamination. However, the direct method has the more vials with more than 60% cell attachment compared to the indirect method mostly in 0–20% cell attachment (Fig. 1D). The direct method used is more effective in optimising the viability of cells after cryopreservation because centrifugation step can cause mechanical stress and disrupt osmotic balance, leading to cell clumping and loss [43]. However, the HDF stored for 1 and 3 months showed no significant differences in terms of the total number of live cells and viability after being revived using both methods (Fig. 2). The cells also retained their elongated and spindle-shaped morphology, indicating that their characteristics and ability to proliferate were maintained after thawing (Figs. 3 and 4). This might be due to the robustness of the fibroblasts and the short storage duration, along with the centrifugal steps.

The storage location can also affect the effectiveness of cryopreservation by exposing cells to different chemical states of liquid nitrogen. Historically, cells fully immersed in liquid nitrogen are more viable because the liquid state is more stable, resulting in less temperature fluctuation compared to vapor or mixed state. This stability reduces the risk of cryo injury, such as cellular dehydration, intracellular crystal formation, and cell death caused by supercooling [44]. Figure 1E and F show that more vials stored in boxes 2 and 3, which are in the vapor phase, have more than 60% cell attachment. With the advent of better-designed storage vessels, storing in the liquid phase has become unnecessary. The vapor phase is preferred because it avoids the risks associated with liquid-phase storage, such as containers potentially exploding when removed, cross-contamination by viruses in the liquid, and exposure of operators to the extremely cold liquid [45]. However, this data does not confirm that vials immersed in liquid nitrogen have the poorest performance. When evaluating frozen storage containers, factors to consider includes the application type (research or clinical), fill volume, temperature, aseptic filling, access for removal, sterility, biocompatibility, potential regulatory requirements, and scalability [30].

Optimising multiple factors during cryopreservation and thawing increases the likelihood of successful cell recovery post-thawing [46]. The evaluation of specific protein expression is essential to characterise and identify if there are any changes in the cryopreserved cells. ICC analysis was performed to evaluate the protein expression of Col-1 and Ki67. Collagen type I is a common protein in the body, making up a large part of bones, ligaments, tendons, and skin which is specific marker that mainly produced by fibroblasts [47,48,49]. Ki-67 helps organise cellular structures during interphase and protects chromosomes during cell division, preventing clumping [50, 51]. By measuring Ki-67 expression, the ability of fibroblast cells to proliferate and remain viable after being frozen and thawed was assessed. The cells without positive Ki67 expression maybe stuck in the G0 or G1 phase of the cell cycle during stabilisation [52, 53]. After 1 and 3 months of cryopreservation, both markers were expressed by HDF (Figs. 5 and 6) in all types of cryo medium with more than 80% for both revival methods which confirm HDF ability to retain its characteristics [1, 54,55,56].

In this study, obtaining data on the type of cryo medium used based on previously collected data posed its own set of challenges, including incomplete records, inconsistencies in the documentation of cryopreservation conditions, and variability in the formulations of cryo medium. Besides, the defective container (Mr cool) resulted in suboptimal cooling rates, and thermal cycling from repeated exposure to varying environmental conditions further stressed the cells. This combination of factors led to reduced cell viability after thawing. Inconsistent handling and storage procedures, transient exposures to non-ideal conditions, and inadequate monitoring of temperature and environmental factors all contributed to compromised cell integrity. Thus, with the advancement of alternative cryoprotective mediums and the meticulous optimisation of storage techniques, cryopreserved cells can maintain their integrity and safety for research and therapeutic use.