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High-throughput proliferation and activation of NK-92MI cell spheroids via a homemade one-step closed bioreactor in pseudostatic cultures for immunocellular therapy

Journal of Biological Engineering volume 18, Article number: 65 (2024) Cite this article

Abstract

The NK-92MI cell line has displayed significant promise in clinical trials for cancer treatment. However, challenges persist in obtaining sufficient cell quantities and achieving optimal cytotoxicity. The proliferation of natural killer (NK) cells involves the formation of cell aggregates, but excessively large aggregates can impede nutrient and waste transport, leading to reduced cell survival rates. In this study, a custom bioreactor was designed to mimic pseudostatic culture conditions by integrating brief mechanical rotation during a 6-h static culture period. This method aimed to achieve an optimal aggregate size while improving cell viability. The findings revealed a 144-fold expansion of 3D NK-92MI cell aggregates, reaching an ideal size of 80–150 µm, significantly increasing both cell proliferation and survival rates. After 14 days of culture, the NK-92MI cells maintained their phenotype during the subsequent phase of cell activation. Moreover, these cells presented elevated levels of IFN-γ expression after IL-18 activation, resulting in enhanced NK cell-mediated cytotoxicity against K562 cells. This innovative strategy, which uses a closed suspension-based culture system, presents a promising approach for improving cell expansion and activation techniques in immunocellular therapy.

Introduction

Conventional cancer treatment typically depends on surgery, chemotherapy, and radiation therapy. However, recent progress in cellular immunotherapy has led to the development of innovative methods involving the manipulation of T cells and natural killer (NK) cells to specifically target cancer surface molecules [1]. This emerging technique has garnered significant interest and shows great potential for cancer treatment.

Traditionally, immunocellular therapy utilizing T cells relies on the patient's own immune cells to prevent graft-versus-host disease [2]. Nevertheless, the autologous approach leads to substantial therapeutic expenses and necessitates the expansion of cell production [3]. In contrast, NK cells, which constitute approximately 10% of circulating lymphocytes, have emerged as a safe alternative for therapy [4, 5]. These cells play a crucial role in the rapid cellular immune response by inhibiting the spread of malignant or infected cells. Furthermore, unlike T cells, NK cells do not require prior activation and can promptly initiate spontaneous killing upon encountering malignant cells. Additionally, they can release cytokines that enhance the function and proliferation of other immune cells in the bloodstream and tumor microenvironment [1].

The collection of NK cells typically involves the gathering of peripheral or cord blood, often requiring the use of specific monoclonal antibodies for precise selection [6]. However, there are concerns about the potential effects of these antibodies on NK cell function and the variability in yield, especially when dysfunctional NK cells are sourced from cancer patients. Furthermore, obtaining a sufficient number of NK cells for therapeutic use requires an in vitro expansion process, which is characterized by significant unpredictability due to the complex behavior of lymphocytes under culture conditions [7,8,9].

An alternative to autologous NK cells is the NK-92 cell line, which originated in the 1990s from mononuclear blood cells taken from patients with aggressive NK-cell lymphoma [10]. The uniformity of the NK-92 cell line offers advantages in terms of engineering feasibility and genetic modification [11, 12]. Additionally, it has shown a favorable safety profile in clinical trials targeting renal, lung, and advanced cancers [13, 14]. While the integration of the NK-92 cell line into various clinical trials has been successful [15,16,17], a growing body of research suggests that achieving effective results in certain cancer types, such as melanoma and lung cancer, may require higher doses (1010 cells) to be not only safe but also necessary for optimal outcomes [13, 14].

Currently, the cultivation of NK cells typically involves static culture methods, often requiring periodic passaging to optimize growth conditions and cell density for faster cell proliferation. In the case of NK-92 cells, their suspension-based growth pattern results in the formation of cellular aggregates [18], which significantly impact cellular behavior, intercellular interactions, and cytokine-mediated signaling within these clusters [19, 20]. In static culture environments, the size of cellular aggregates typically ranges from 200–500 μm [18]. However, larger aggregates may lead to apoptosis in core cells due to limited oxygen and nutrient diffusion, underscoring the importance of achieving an appropriate aggregate size [18, 21]. Nonetheless, studies have shown that NK-92 cells can produce increased quantities of extracellular vesicles through mechanical stimulation in a specialized seesaw-motion bioreactor designed for cancer therapy [22].

To address the challenge of cell aggregation and leverage the benefits of dynamic culture environments to increase therapeutic effectiveness, we created a custom bioreactor that mimics pseudostatic culture conditions. This method involves brief mechanical rotation during a 6-h static culture period, with the goal of achieving an optimal cell aggregate size and improving cell viability. This approach eliminates the need for additional passaging, reduces cell loss, and minimizes cultivation disruption. Moreover, we employed NK-92MI cells, a modified IL-2-independent NK-92 cell line known for enhancing immune responses against cancer. To further increase the cytotoxic potential of NK-92MI cells in targeting tumor cells, we introduced a second centrifuge tube into a homemade bioreactor loaded with IL-15 or IL-18. The cells initially multiplied in the upper tube until a specific cell count was reached. Using two-way valves, the proliferated NK-92MI cells were subsequently transferred to the lower tube for activation. We anticipate that NK-92MI cells will be activated, influencing gene expression and demonstrating improved tumor-cell-killing abilities. Compared with conventional NK cell culture methods, the bioreactor system in our study not only addresses apoptosis related to NK cell aggregation but also supports the long-term cultivation and activation of NK cells within a closed system to prevent contamination and ensure user-friendly operation. Furthermore, this approach can be adapted for various immune cells, promoting their growth and activation and offering a high-throughput immune cell culture system for immunocellular therapy. The study design is depicted in Scheme 1.

Scheme 1.
scheme 1

Schematic illustration of a homemade bioreactor that simulates pseudostatic culture conditions for NK-92 MI cell expansion and activation

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Results & discussion

Proliferation of NK-92MI cells

To assess the impact of different rotation parameters on overall cell proliferation, various rotation cycles were initially measured (Fig. 1). The cells cultured via traditional methods in static dishes exhibited rapid growth during the initial 7 d. The cells then stabilized and exhibited slow growth, reaching an approximately 84-fold increase in cell number by day 14. This deceleration resulted from the formation of larger cell aggregates, which, in turn, reduced the cell proliferation rate in the later stages of static culture. In contrast, the pseudostatic culture showed relatively slower growth in the first 7 d but grew faster in the subsequent period. This is due to an adaptation phase in the pseudostatic bioreactor during the initial 7 d, during which period rotation assists in optimizing cell aggregation and dispersion. The optimal cell aggregate size significantly affects cell‒cell interactions, increasing the availability of crucial soluble factors, such as growth factors, cytokines, nutrients, and bioactive molecules, which are important for mediating cellular proliferation [23]. In the pseudostatic culture system, an inadequate rotation frequency (15 min/24 h) resulted in suboptimal dispersion, whereas an excessively high frequency (15 min/3 h) affected cell aggregation, consequently impeding proliferation. At an appropriate rotation frequency (15 min/6 h), the cells showed a remarkable 144-fold increase in proliferation by day 14, significantly increasing their potential for cell expansion compared with that of conventional static dish cultures.

Fig. 1
figure 1

The proliferation of NK-92MI cells cultured in a static dish control system and a pseudostatic bioreactor system on days 4, 7, 11, and 14

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Shear stress has also been identified as a critical factor in maintaining cell cultures in bioreactors [24]. The gentle agitation generated by the pseudostatic system's rotation not only promotes uniform distribution of nutrients and oxygen but also applies controlled shear stress to the cells. This helps regulate aggregate size and structure, preventing the formation of overly large clusters that could impede mass transfer. By fine-tuning the rotation parameters, the bioreactor achieves a balance between promoting cell growth and avoiding excessive stress, which could otherwise lead to cell damage or death.

Viability of NK-92MI cells

The viability of NK-92MI cells under various culture conditions was assessed by flow cytometry. Although the total cell count was greater after 7 d of static culture (Fig. 1), the increase in cell aggregation and local accumulation of waste slightly reduced cell viability compared with that of the other pseudostatic groups, resulting in approximately 65% viability after 14 d (Fig. 2). In the bioreactor group employing periodic rotation, where the size of the cell aggregates affects the proliferation efficiency, the cell viability consistently surpassed 85% after 14 d. These findings underscore the need for suitable dynamic culture conditions to maintain NK-92MI cell survival. To optimize cell expansion and viability, the parameters were standardized to a rotation of 15 min/6 h at 15 rpm to ensure the most effective cell amplification ratio and sustained cell viability.

Fig. 2
figure 2

The viability of NK-92MI cells cultured in a static dish control system and a pseudostatic bioreactor system was evaluated via flow cytometry on days 4, 7, 11, and 14

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Live/dead cell staining and NK-92MI cell aggregate size

After 1, 7, and 14 d of cell culture in both static dishes and bioreactors, meticulously isolated cell aggregates were observed via confocal microscopy to perform live/dead cell staining (Fig. 3). The average aggregate sizes are summarized in Table 1. In static culture, the sizes of the cell aggregates on days 7 and 14 were 140.71 ± 48.97 µm and 395.70 ± 86.15 µm, respectively. Conversely, the periodic shear stress of the bioreactor’s pseudostatic culture notably decreased the cell aggregate size, which averaged approximately 118.44 ± 32.13 µm by day 14. Such variations in aggregate size directly affect survival rates. Although NK-92MI cells cultured in traditional static culture initially grew rapidly (Fig. 1), after 14 days, the inner part of the larger cell mass underwent apoptosis owing to limited mass transfer (Fig. 3). In contrast, the bioreactor group with an appropriate cell aggregate size exhibited fewer instances of apoptosis after 14 days, as depicted in Fig. 2.

Fig. 3
figure 3

Evaluation of NK-92MI cells via a live/dead cell staining assay after 1–7 and 14 days of cultivation. Living cells in green were stained with calcein AM, and dead cells in red were stained with propidium iodide. Scale bar = 100 μm

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Table 1 The Average ± standard deviation of cell aggregate size in static dish and pseudo-static bioreactor systems
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When the cell aggregate size exceeds 300 µm, nutrient and oxygen transport to and waste transport from the center of the aggregates become suboptimal, leading to apoptosis in the central cells of the aggregates [23,24,25,26]. This finding was corroborated by live/dead cell staining after analysis via flow cytometry (Fig. 4). While a certain level of cell death is expected in culture, the data clearly show that the pseudostatic culture method significantly reduces the proportion of dead cells compared to static culture conditions. And Fig. 5 illustrates the general size distribution, indicating that the bioreactor system effectively maintained size control.

These results suggest that the homemade bioreactor system developed in this study effectively generated cell aggregates of appropriate size, promoted cell proliferation and mass transfer, and ultimately improved cell survival rates.

Fig. 4
figure 4

Flow cytometry analysis of the live/dead stained NK-92 MI cells after 14 d of cultivation. a Cells were cultured via a traditional static culture method in a culture dish. b Cells were cultured via a pseudostatic culture method in a custom pseudostatic bioreactor

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Fig. 5
figure 5

Observation of spheroid morphology and size distribution under static and pseudostatic culture methods after 14 days of cultivation. Bright field images in (a) show the morphological characteristics of cell spheroids in static dish cultures, while (c) illustrates those in a bioreactor system. The size distributions for the static and pseudostatic culture methods are presented in (b) and (d), respectively. Scale bar = 100 μm

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Phenotypic changes in activated NK-92MI cells in the self-designed bioreactor

After optimizing the pseudostatic culture of NK-92MI cells, they were further introduced into a self-designed, one-step, homemade bioreactor system, allowing the cells to proliferate within a closed system and undergo IL-15- or IL-18-induced activation. To explore potential alterations in the NK-92MI cell phenotype induced by this culture system, qPCR analysis was performed to measure several NK-92MI cell markers (Fig. 6). The mRNA levels of the positive markers CD2 and CD56 were detected in NK-92MI cells, whereas the native negative marker CD16 was not detected [15, 27]. These findings suggested that neither the bioreactor culture system nor activation with IL-15 or IL-18 significantly altered the specific phenotype of NK-92MI cells.

Fig. 6
figure 6

Positive NK-92MI markers, including CD2 and CD56, and the negative NK-92MI marker CD16 were used to identify the NK-92MI phenotype. The mRNA levels are expressed as relative levels normalized to the GAPDH level (defined as 1)

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Cytotoxicity-related gene expression in NK-92MI cells after homemade bioreactor cultivation

We further investigated the genes associated with immunotherapy and cancer cell killing in NK-92MI cells and compared their expression levels with those of cells cultured conventionally in cell dishes (Fig. 7). NKG2A is an inhibitory NK receptor that recognizes specific ligands on target cells and transmits inhibitory signals [28]. Moreover, NK cells possess the dual capacity to produce proinflammatory (IFN-γ) and anti-inflammatory (IL-10) cytokines, highlighting their essential role both as inflammatory agents for clearing infections and as regulators to manage inflammation and mitigate immune-mediated damage to the host [29, 30]. Higher expression levels of IFN-γ in NK-92MI cells correlate with a stronger ability to kill cancer cells, while IL-10 acts as an antagonist of NK cell activation [31, 32]. Compared with those of normal NK-92MI cells, upon entry into the reactor, the expression levels of genes associated with the inhibition of NK-92MI cell activation, such as NKG2A and IL-10, were slightly decreased. Conversely, the expression levels of genes related to activated NK-92MI cells, particularly IFN-γ, were significantly increased, especially in the IL-15 and IL-18 activation groups. Compared with those in normal NK-92MI cells, the gene expression levels of IFN-γ in the IL-15 and IL-18 activation groups were upregulated 2.8- and 3.2-fold, respectively. These findings are consistent with those of previous studies that utilized IL-15 or IL-18 for NK cell activation [32, 33].

Fig. 7
figure 7

Cytotoxicity-related genes, including NKG2A, IFN-γ, and IL-10, were used to evaluate the increase in gene expression in activated NK-92MI cells. Relative gene expression levels were normalized to GAPDH gene expression levels and expressed as the fold change compared with the levels in normal NK-92MI cells

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NK-92MI cell-mediated cellular cytotoxicity

To determine the activity of NK-92MI cells against cancer cells via the developed homemade one-step proliferation and activation closed bioreactor culture system, the human erythroleukemic cell line K562 was used as the target cell line (Fig. 8).

Fig. 8
figure 8

NK-92MI cell-mediated cytotoxicity against K562 cells. After the proliferation and activation of NK-92MI cells induced by IL-18 through a homemade bioreactor, the cells showed a cytotoxic effect on K562 cells after coculture for 0 h (a), 2 h (b), 3 h (c), and 4 h (d). e Quantification of NK-92MI cell-mediated cytotoxicity was determined by measuring the fluorescence intensity in the supernatant via a plate reader

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After labeling with calcein AM, K562 cells exhibited green fluorescence under a fluorescence microscope. After 14 days of proliferation and activation, the NK-92MI cells were cultured. As shown in Fig. 8b–d, within 4 h, the amount of fluorescent dye released from the killed K562 cells gradually decreased, reducing the fluorescence intensity of the cells. The real-time behavior of NK-92MI cells in response to the target K562 cancer cells is shown in Supplementary Video S1. These outcomes enabled further collection of supernatants and the quantitative assessment of cytotoxicity via a specific lysis formula [2, 34]. The results confirmed that the activated cells exhibited enhanced cytotoxicity against K562 cells (Fig. 8e). Specifically, the IL-18-activated group exhibited a 1.62-fold increase in cytotoxicity compared with the nonactivated group. Previous studies have indicated that the cytotoxicity of NK-92MI cells to cancer cells can be increased through IL-18-induced activation [33, 35]. However, IL-15-induced NK-92MI cell-mediated cytotoxicity was not notably pronounced, with a 1.17-fold increase (Fig. 8). It has been reported that IL-15 has some influence on cell survival and can enhance cellular cytotoxicity within a short 24 h period, but this effect diminishes after 96 h [35]. In this study, the relatively modest impact of IL-15 activation on cytotoxicity may be attributed to its suitability for the short-term activation of NK-92 cells.

It has been reported that cytokines captured by NK cells can indirectly trigger signal transduction in surrounding NK cells through cell‒cell interactions within the cell aggregate, maximizing the utilization of cytokines [18, 36]. Therefore, maintenance of aggregate characteristics may be directly related to cell functionality. Additionally, providing adequate mechanical stimulation, including turbulence and stress to NK-92MI cells, would activate NK-92MI cells and generate functional extracellular vesicles of high quality and quantity for cancer treatment [22].

In this study, we designed a custom one-step proliferation and activation closed bioreactor culture system. The NK-92MI cells rapidly proliferated in the upper chamber, forming the adequate cell aggregate size necessary for the desired cell count and survival. Through simple valve control, the 3D NK-92MI cell aggregates were subsequently directed to the lower chamber for specific activation in a single step, avoiding cell loss and cultivation interference during the 14-d culture period. While their distinct cell surface phenotypes are maintained, activated cells enhance the expression of genes associated with cell cytotoxicity, thereby increasing NK-92MI cell-mediated cytotoxicity against tumor cells. This closed system ensures high efficiency, is eco-friendly, is easy to operate, and mitigates the overall experimental contamination risk. Moreover, this design has potential for broader applications in the proliferation and activation of other suspended immune cells, potentially opening new avenues for immunocellular therapy.

Conclusions

This study details the successful development of a homemade, one-step proliferation and activation closed bioreactor culture system. This system facilitates brief mechanical rotation of 3D NK-92MI cell aggregates during a 6-h static culture period. The expansion of NK-92MI cells reached 144-fold, approximately 1.8 times greater than that in the static control culture system. This approach resulted in the attainment of an optimal cell aggregate size (80–150 µm), leading to improved cell proliferation and survival rates in a pseudostatic culture system. Furthermore, the NK-92MI cells maintained their phenotypes after 14 days of culture and exhibited increased IFN-γ expression levels following IL-18 activation. Notably, the one-step proliferation and activation of NK-92MI cells increased NK cell-mediated cytotoxicity against K562 cells by 1.6-fold compared with that of normal NK-92MI cells. These findings underscore the potential of the homemade functionally closed bioreactor system as a safe and cost-effective tool for immunocellular therapy.

Materials and methods

Materials

NK-92MI cells were purchased from ATCC (Manassas, VA, USA). K562 cells were purchased from BCRC (Hsinchu, Taiwan). Minimum essential medium eagle alpha modification (α-MEM), Iscove's modified Dulbecco's medium (IMDM), phosphate-buffered saline (PBS), calcein AM, propidium iodide, and TRIzol reagent were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Inositol and folic acid were purchased from Sigma‒Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and horse serum (HS) were purchased from HyClone (Logan, Utah, USA). The primary antibiotic was obtained from InvivoGen (San Diego, CA, USA). The Direct-zol™ RNA MiniPrep Kit was purchased from Zymo Research (Irvine, CA, USA). The KAPA SYBR® FAST One-Step qRT‒PCR Kit was obtained from KAPA Biosystems (Wilmington, MA, USA).

Expansion of NK-92 cells

The complete NK culture medium for NK-92MI cells was composed of α-MEM supplemented with 0.2 mM inositol, 0.02 mM folic acid, 12.5% HS, 12.5% FBS, and 0.1% primary antibiotic. NK-92MI cells were cultured in 50 mL centrifuge tubes with a 0.22 μm filter on the cap. Approximately 100,000 NK-92MI cells in 10 mL of complete culture medium were added to a tube and placed on a tube roller (MX-T6-S; DLAB, Beijing, China). The cells were cultured under different pseudostatic culture conditions of brief mechanical rotation at 15 rpm for 15 min after every 3, 6, or 24 h of static culture. They were cultured for 14 d, followed by the addition of 5 mL of medium every 3 d. Cells cultured in a normal Petri dish served as controls.

Flow cytometry analysis of NK-92 cells

The proliferation and viability of NK-92MI cells cultured in the homemade bioreactor were assessed via flow cytometry on days 1, 4, 7, 11, and 14. First, the cells were collected, washed with PBS and stained with 0.1 μM calcein AM and 3 μM propidium iodide for 15 min at room temperature in the dark. The total and live cells were counted via a flow cytometer (Attune NxT; Invitrogen, Carlsbad, CA, USA).

Live/dead NK-92MI cell staining

To measure the NK-92MI cell aggregate size and the degree of cell necrosis, the cells were collected on days 1, 7, and 14; washed with PBS; and stained with 2 μM calcein AM and 1 μM propidium iodide for 15 min at room temperature in the dark. The microspheres were observed under a confocal microscope (LSM-900; Zeiss, Oberkochen, Germany). Living and dead cells are shown in green and red, respectively, on the basis of the specific wavelength of light excitation.

Proliferation and activation of NK-92MI cells in a self-designed bioreactor

NK-92MI cells were cultured in a custom-designed bioreactor consisting of two parts: a 50 mL centrifuge tube with a 0.22 μm filter as the upper tube and a normal 50 mL centrifuge tube as the lower tube. The upper and lower tubes were connected by a two-way valve by drilling a hole with a diameter of 6 mm and were attached via a hot-melt adhesive. Approximately 100,000 NK92-MI cells in 30 mL of complete culture medium were placed in the upper tube, and the lower tube was loaded with 1 mL of 0.9 μg/mL IL-15 or IL-18. The cells were cultured under pseudostatic culture conditions with brief mechanical rotation at 15 rpm for 15 min during a 6-h static culture period for 7 d. On day 8, the cells flowed from the upper tube to the lower tube through the two-way valve and continued culturing under pseudodynamic culture conditions with brief mechanical rotation at 15 rpm for 15 min during a 6-h static culture period for another 7 d.

Gene expression in NK-92MI cells

On day 14, after the NK-92MI cells were cultured in a custom-designed bioreactor, they were washed with PBS and lysed with TRIzol reagent. The supernatant was collected, and total RNA was extracted via the Direct-zol™ RNA MiniPrep Kit following the manufacturer’s protocol. The RNA samples were analyzed via quantitative polymerase chain reaction (qPCR) via the KAPA SYBR® FAST One-Step qRT‒PCR Kit. The primers used for this analysis are listed in Supplementary Table S1. The intensities were detected and recorded via a ViiA 7 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). The mRNA levels of CD2, CD16, and CD56 were calculated via the comparative Ct method [25], with GAPDH used as the reference to calculate relative mRNA levels. Moreover, cytotoxic genes, including IL-10, INF-γ, and NKG2A, were used to evaluate the cytotoxicity of NK-92MI cells. Relative gene expression levels were determined after normalization to GAPDH gene expression levels and are expressed as the fold change relative to the control group. NK-92 cells cultured in a normal Petri dish served as controls.

NK-92MI cell-mediated cytotoxicity

NK cell-mediated cytotoxicity was determined via a calcein AM-labeled K562 cytotoxicity assay. First, K562 cells were stained with 10 µM calcein AM for 30 min and then washed three times with IMDM. Approximately 50,000 NK-92MI cells suspended in 100 μL of complete culture medium obtained from the bioreactor were cocultured with 50,000 calcein AM-labeled K562 cells in 100 μL of IMDM in a round-bottom 96-well plate at 37 °C with 5% CO2 for 4 h. Real-time images of NK-92MI cells interacting with K562 tumor cells were captured via the autorecording system of the LSM-900 confocal microscope at a rate of 30 fps. After incubation, the 96-well plate was centrifuged at 120 × g for 1 min, and the supernatants (100 μL each) were transferred to a new flat-bottom black 96-well plate for detection. At the end of the reactions, the fluorescence was read in a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The spontaneously released samples were calcein AM-stained target cells (K562) without effector cells (NK-92MI). The maximum-release samples were stained with Triton X-100. Cytotoxicity was calculated as follows: \({\%\;\mathrm{dead}\;\mathrm{tumor}\;\mathrm{cells}=}\frac{\mathrm{Experimental}\;\mathrm{cell}\;\mathrm{death}\;-\;\mathrm{Spontaneous}\;\mathrm{cell}\;\mathrm{death}\;}{\mathrm{Maximum}\;\mathrm{cell}\;\mathrm{death}\;-\;\mathrm{Spontaneous}\;\mathrm{cell}\;\mathrm{death}}\).

Statistics

The results obtained in this study are expressed as the means and standard deviations of at least three replicates. One-way analysis of variance with multiple comparisons and Student’s t tests were used for all the statistical analyses. Differences were considered statistically significant at p values less than 0.05. (p < 0.05, ∗ ; p < 0.01, ∗ ∗ ; p < 0.001, ∗ ∗ ∗).

Data availability

No datasets were generated or analysed during the current study.

References

  1. Klingemann H. The NK-92 cell line—30 years later: its impact on natural killer cell research and treatment of cancer. Cytotherapy. 2023;25(5):451–7.

    Article  Google Scholar 

  2. Shin HY, Jang S, Woo HJ, Chung J-H, Kim W-H, Kim D, Kang M, Lim Y, Habib O, Lee J. Cytokine engineered NK-92 therapy to improve persistence and anti-tumor activity. Theranostics. 2023;13(5):1506.

    Article  Google Scholar 

  3. Yang Y, Jacoby E, Fry TJ. Challenges and opportunities of allogeneic donor-derived CAR T cells. Curr Opin Hematol. 2015;22(6):509.

    Article  Google Scholar 

  4. Klingemann H, Boissel L, Toneguzzo F. Natural killer cells for immunotherapy–advantages of the NK-92 cell line over blood NK cells. Front Immunol. 2016;7:91.

    Article  Google Scholar 

  5. Myers JA, Miller JS. Exploring the NK cell platform for cancer immunotherapy. Nat Rev Clin Oncol. 2021;18(2):85–100.

    Article  Google Scholar 

  6. Kundu S, Gurney M, O’Dwyer M. Generating natural killer cells for adoptive transfer: expanding horizons. Cytotherapy. 2021;23(7):559–66.

    Article  Google Scholar 

  7. Harada H, Saijo K, Watanabe S, Tsuboi K, Nose T, Ishiwata I, Ohno T. Selective expansion of human natural killer cells from peripheral blood mononuclear cells by the cell line, HFWT. Jpn J Cancer Res. 2002;93(3):313–9.

    Article  Google Scholar 

  8. Masuyama J-I, Murakami T, Iwamoto S, Fujita S. Ex vivo expansion of natural killer cells from human peripheral blood mononuclear cells costimulated with anti-CD3 and anti-CD52 monoclonal antibodies. Cytotherapy. 2016;18(1):80–90.

    Article  Google Scholar 

  9. Damodharan SN, Walker KL, Forsberg MH, McDowell KA, Bouchlaka MN, Drier DA, Sondel PM, DeSantes KB, Capitini CM. Analysis of ex vivo expanded and activated clinical-grade human NK cells after cryopreservation. Cytotherapy. 2020;22(8):450–7.

    Article  Google Scholar 

  10. Gong J-H, Maki G, Klingemann HG. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia. 1994;8(4):652–8.

    Google Scholar 

  11. Liu H, Yang B, Sun T, Lin L, Hu Y, Deng M, Yang J, Liu T, Li J, Sun S. Specific growth inhibition of ErbB2-expressing human breast cancer cells by genetically modified NK-92 cells. Oncol Rep. 2015;33(1):95–102.

    Article  Google Scholar 

  12. Nowakowska P, Romanski A, Miller N, Odendahl M, Bonig H, Zhang C, Seifried E, Wels WS, Tonn T. Clinical grade manufacturing of genetically modified, CAR-expressing NK-92 cells for the treatment of ErbB2-positive malignancies. Cancer Immunol Immunother. 2018;67:25–38.

    Article  Google Scholar 

  13. Arai S, Meagher R, Swearingen M, Myint H, Rich E, Martinson J, Klingemann H. Infusion of the allogeneic cell line NK-92 in patients with advanced renal cell cancer or melanoma: a phase I trial. Cytotherapy. 2008;10(6):625–32.

    Article  Google Scholar 

  14. Tonn T, Schwabe D, Klingemann HG, Becker S, Esser R, Koehl U, Suttorp M, Seifried E, Ottmann OG, Bug G. Treatment of patients with advanced cancer with the natural killer cell line NK-92. Cytotherapy. 2013;15(12):1563–70.

    Article  Google Scholar 

  15. Suck G, Odendahl M, Nowakowska P, Seidl C, Wels WS, Klingemann HG, Tonn T. NK-92: an ‘off-the-shelf therapeutic’for adoptive natural killer cell-based cancer immunotherapy. Cancer Immunol Immunother. 2016;65:485–92.

    Article  Google Scholar 

  16. Törnroos H, Hägerstrand H, Lindqvist C. Culturing the human natural killer cell line NK-92 in interleukin-2 and interleukin-15–Implications for clinical trials. Anticancer Res. 2019;39(1):107–12.

    Article  Google Scholar 

  17. Zhang C, Oberoi P, Oelsner S, Waldmann A, Lindner A, Tonn T, Wels WS. Chimeric antigen receptor-engineered NK-92 cells: an off-the-shelf cellular therapeutic for targeted elimination of cancer cells and induction of protective antitumor immunity. Front Immunol. 2017;8: 533.

    Article  Google Scholar 

  18. Aponte OFG, Kozma B, Egger D, Kasper C, Herwig C. Kinetics of NK-92 growth and functionality in pseudostatic cultures. Biochem Eng J. 2023;196: 108929.

    Article  Google Scholar 

  19. Kim M, Kim T-J, Kim HM, Doh J, Lee K-M. Multicellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster. Sci Rep. 2017;7(1):40623.

    Article  Google Scholar 

  20. Lee K-M, Forman JP, McNerney ME, Stepp S, Kuppireddi S, Guzior D, Latchman YE, Sayegh MH, Yagita H, Park C-K. Requirement of homotypic NK-cell interactions through 2B4 (CD244)/CD48 in the generation of NK effector functions. Blood. 2006;107(8):3181–8.

    Article  Google Scholar 

  21. Ding H, George S, Leng XI, Ihnat M, Ma J-X, Jiang G, Margolis D, Dumond J, Zhang Y. Silk fibers assisted long-term 3D culture of human primary urinary stem cells via inhibition of senescence-associated genes: potential use in the assessment of chronic mitochondrial toxicity. Materia Today Advances. 2022;15: 100261.

    Article  Google Scholar 

  22. Wu J, Wu D, Wu G, Bei H-P, Li Z, Xu H, Wang Y, Wu D, Liu H, Shi S. Scale-out production of extracellular vesicles derived from natural killer cells via mechanical stimulation in a seesaw-motion bioreactor for cancer therapy. Biofabrication. 2022;14(4): 045004.

    Article  Google Scholar 

  23. Chen C-Y, Ke C-J, Yen K-C, Hsieh H-C, Sun J-S, Lin F-H. 3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy. Theranostics. 2015;5(6):643.

    Article  Google Scholar 

  24. Fernanda P, Marcelino PRF, Schlogl AE, José ÁHM, Rodrigues RdCLB, Fabrino DL, Santos IJB, Rosa CA. Bioreactors: applications and Innovations for a sustainable and healthy future—a critical review. Appl Sci. 2024;14(20):9346.

    Article  Google Scholar 

  25. Yang I-H, Kuan C-Y, Chen Z-Y, Li C-H, Chi C-Y, Lin Y-Y, Liang Y-J, Kuo W-T, Li Y-A, Lin F-H. Engineered cell-laden thermosensitive poly (N-isopropylacrylamide)-immobilized gelatin microspheres as 3D cell carriers for regenerative medicine. Materials Today, Bio. 2022;15: 100266.

    Article  Google Scholar 

  26. Yang I-H, Chen Y-S, Li J-J, Liang Y-J, Lin T-C, Jakfar S, Thacker M, Wu S-C, Lin F-H. The development of laminin-alginate microspheres encapsulated with Ginsenoside Rg1 and ADSCs for breast reconstruction after lumpectomy. Bioactive Materials. 2021;6(6):1699–710.

    Article  Google Scholar 

  27. Briones JC, Espulgar WV, Koyama S, Yoshikawa H, Park J, Naito Y, Kumanogoh A, Tamiya E, Takamatsu H, Saito M. A microfluidic platform for single cell fluorometric granzyme B profiling. Theranostics. 2020;10(1):123.

    Article  Google Scholar 

  28. Lanier LL. Up on the tightrope: natural killer cell activation and inhibition. Nat Immunol. 2008;9(5):495–502.

    Article  Google Scholar 

  29. Martinez-Espinosa I, Serrato JA, Ortiz-Quintero B. Role of IL-10-producing natural killer cells in the regulatory mechanisms of inflammation during systemic infection. Biomolecules. 2021;12(1): 4.

    Article  Google Scholar 

  30. Lima BV, Oliveira MJ, Barbosa MA, Goncalves RM, Castro F. Harnessing chitosan and poly-(γ-glutamic acid)-based biomaterials toward cancer immunotherapy. Materials Today, Advances. 2022;15: 100252.

    Article  Google Scholar 

  31. Thomas H, Jäger M, Mauel K, Brandau S, Lask S, Flohé SB. Interaction with mesenchymal stem cells provokes natural killer cells for enhanced IL-12/IL-18-induced interferon-gamma secretion. Mediators Inflamm. 2014;2014(1):143463.

    Google Scholar 

  32. Park JY, Lee SH, Yoon S-R, Park Y-J, Jung H, Kim T-D, Choi I. IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells. Mol Cells. 2011;32:265–72.

    Article  Google Scholar 

  33. Choi YH, Lim EJ, Kim SW, Moon YW, Park KS, An H-J. IL-27 enhances IL-15/IL-18-mediated activation of human natural killer cells. J Immunother Cancer. 2019;7:1–12.

    Google Scholar 

  34. Yang C, Du M, Yan F, Chen Z. Focused ultrasound improves NK-92MI cells infiltration into tumors. Front Pharmacol. 2019;10: 326.

    Article  Google Scholar 

  35. Mikhailova V, Khokhlova E, Grebenkina P, Salloum Z, Nikolaenkov I, Markova K, Davidova A, Selkov S, Sokolov D. NK-92 cells change their phenotype and function when cocultured with IL-15, IL-18 and trophoblast cells. Immunobiology. 2021;226(5): 152125.

    Article  Google Scholar 

  36. Kim D, Jo S, Lee D, Kim S-M, Seok JM, Yeo SJ, Lee JH, Lee JJ, Lee K, Kim T-D. NK cells encapsulated in micro/macropore-forming hydrogels via 3D bioprinting for tumor immunotherapy. Biomat Res. 2023;27(1):1–16.

    Article  Google Scholar 

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Acknowledgements

The study was supported by the National Health Research Institutes, Taiwan (BN-113-PP-01, BN-113-GP-09.) and subsidized by Ministry of Science and Technology and National Taiwan University (NTU), Taiwan.

Funding

This work was funded by the National Health Research Institutes, Taiwan (BN-113-PP-01, BN-113-GP-09.)

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Authors and Affiliations

  1. Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, No. 35, Keyan Road, Zhunan, Miaoli, 35053, Taiwan

    Jhih-Ni Lin, Che-Yung Kuan, Chia-Ting Chang, Zhi-Yu Chen, Yu-Ying Lin, I.-Hsuan Yang & Feng-Huei Lin

  2. Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 49, Fanglan Rd, Taipei, 10672, Taiwan

    Jhih-Ni Lin, Che-Yung Kuan, Zhi-Yu Chen, Wei-Ting Kuo, Jason Lin, I.-Hsuan Yang & Feng-Huei Lin

  3. National Chung Hsing University, Taichung, Taiwan

    Chia-Ting Chang & Yu-Ying Lin

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  1. Jhih-Ni Lin
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Contributions

Jhih-Ni Lin. wrote the main manuscript text and conducted the methodology, formal analysis, investigation, visualization, and wrote the original draft. Chia-Ting Chang, Che-Yung Kuan, Zhi-Yu Chen, and Wei-Ting Kuo contributed to the methodology, investigation, and validation. Feng-Huei Lin and I-Hsuan Yang handled conceptualization, methodology, formal analysis, funding acquisition, and supervised the writing—review & editing. All authors reviewed the manuscript.

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Correspondence to I.-Hsuan Yang or Feng-Huei Lin.

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Lin, JN., Kuan, CY., Chang, CT. et al. High-throughput proliferation and activation of NK-92MI cell spheroids via a homemade one-step closed bioreactor in pseudostatic cultures for immunocellular therapy. J Biol Eng 18, 65 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13036-024-00461-0

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Journal of Biological Engineering

ISSN: 1754-1611