Enhanced production of recombinant coxsackievirus A16 using a serum-free HEK293A suspension culture system for bivalent enterovirus vaccine development.

Coxsackievirus A16 (CVA16) is one of the primary pathogens that causes hand, foot, and mouth disease (HFMD) in young children. In previous studies, CVA16 vaccine development has encountered several challenges, such as inefficient replication of the CVA16 virus in present culture systems, the induction of only mild neutralizing antibody titers, and neutralizing antibodies induced by certain vaccine candidates that are unable to protect against CVA16 viral challenge. In this study, we constructed a DNA-launched CVA16 infectious clone (CVA16ic) based on the genomic sequence of the CVA16 N5079 strain to minimize interference from viral quasispecies. The biochemical properties of this CVA16ic strain were similar to those of its parental strain. Serum-free HEK293A suspension cells, which produced higher virus titers than Vero cells, were demonstrated to improve CVA16 production yields. In addition, our study showed that inactivated EV-A71 antigens could enhance the immunogenicity of inactivated CVA16 mature/full particles (F-particles), suggesting that a bivalent CVA16 and EV-A71 vaccine may be an effective strategy for CVA16 vaccine development. These findings are expected to provide novel strategies and accelerate the development of bivalent HFMD vaccines.

Comparative Analysis of Serum and Serum-Free Medium Cultured Mesenchymal Stromal Cells for Cartilage Repair.

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium's ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair.

Serum-free medium for recombinant protein expression in insect cells.

The baculovirus expression vector system (BEVS) has been widely used to produce recombinant proteins because of several advantages, such as eukaryotic post-translational modifications similar to those in mammalian cells, high expression levels and safety, and large gene capacity. Usually, insect cell culture requires 5%‒10% fetal bovine serum, which has many adverse effects, including high cost, heterogeneity between batches, complex composition, and pollution risks. Therefore, serum-free medium (SFM) is indispensable for the production of recombinant proteins in insect cell culture. Here, the most commonly used insect cell lines and three insect cell media, namely basic medium, SFM, and chemically defined medium, are summarized. The basic components of insect cell SFM are similar to those of other cells but contain special components. The components, functions, and issues of different SFM used for insect cell culture are reviewed. In recent years, some special additives have been demonstrated to increase recombinant protein expression yield and quality in BEVS, and the functions and possible mechanisms of small-molecule additives are reviewed herein. Finally, future perspectives of SFM used in BEVS for recombinant protein production are discussed.

Serum-Free Media Formulation Using Marine Microalgae Extracts and Growth Factor Cocktails for Madin-Darby Canine Kidney and Vero Cell Cultures.

The development of serum-free media (SFM) is critical to advance cell culture techniques used in viral vaccine production and address the ethical concerns and contamination risks associated with fetal bovine serum (FBS). This study evaluated the effects of marine microalgal extracts and growth factor cocktails on the activity of Madin-Darby canine kidney (MDCK) and Vero cells. Five marine microalgal species were used: Spirulina platensis (SP), Dunaliella salina (DS), Haematococcus pluvialis (HP), Nannochloropsis salina (NS), and Tetraselmis sp. (TS). DS and SP extracts significantly increased the proliferation rate of both MDCK and Vero cells. DS had a proliferation rate of 149.56% and 195.50% in MDCK and Vero cells, respectively, compared with that in serum-free medium (SFM). Notably, DS and SP extracts significantly increased superoxide dismutase (SOD) activity, which was 118.61% in MDCK cells and 130.08% in Vero cells for DS, and 108.72% in MDCK cells and 125.63% in Vero cells for SP, indicating a reduction in intracellular oxidative stress. Marine microalgal extracts, especially DS and SP, are feasible alternatives to FBS in cell culture as they promote cell proliferation, ensure safety, and supply essential nutrients while reducing oxidative stress.

Next generation yellow fever vaccine induces an equivalent immune and transcriptomic profile to the current vaccine: observations from a phase I randomised clinical trial.

BACKGROUND: Yellow fever (YF), a mosquito-borne acute viral haemorrhagic illness, is endemic to many tropical and subtropical areas of Africa and Central and South America. Vaccination remains the most effective prevention strategy; however, as repeated outbreaks have exhausted vaccine stockpiles, there is a need for improved YF vaccines to meet global demand. A live-attenuated YF vaccine candidate (referred to as vYF) cloned from a YF-17D vaccine (YF-VAX®) sub-strain, adapted for growth in Vero cells cultured in serum-free media, is in clinical development. We report the innate and adaptive immune responses and the transcriptome profile of selected genes induced by vYF. METHODS: Healthy adults aged 18-60 years were randomised at a 1:1:1:1 ratio to receive one dose of vYF at 4, 5 or 6 Log CCID50 or YF-VAX (reference vaccine), administered subcutaneously in the upper arm (ClinicalTrials.gov identifier: NCT04142086). Blood/serum samples were obtained at scheduled time points through 180 days (D180) post-vaccination. The surrogate endpoints assessed were: serum cytokine/chemokine concentrations, measured by bead-based Multiplex assay; peripheral blood vYF-specific IgG and IgM memory B cell frequencies, measured by FluoroSpot assay; and expression of genes involved in the immune response to YF-17D vaccination by RT-qPCR. FINDINGS: There was no increase in any of the cytokine/chemokine concentrations assessed through D14 following vaccination with vYF or YF-VAX, except for a slight increase in IP-10 (CXCL10) levels. The gene expression profiles and kinetics following vaccination with vYF and YF-VAX were similar, inclusive of innate (antiviral responses [type-1 interferon, IFN signal transduction; interferon-stimulated genes], activated dendritic cells, viral sensing pattern recognition receptors) and adaptive (cell division in stimulated CD4+ T cells, B cell and antibody) immune signatures, which peaked at D7 and D14, respectively. Increases in vYF-specific IgG and IgM memory B cell frequencies at D28 and D180 were similar across the study groups. INTERPRETATION: vYF-induced strong innate and adaptive immune responses comparable to those induced by YF-VAX, with similar transcriptomic and kinetic profiles observed. FUNDING: Sanofi.

Non-Specific Elevated Serum Free Fatty Acids in Lung Cancer Patients: Nutritional or Pathological?

IMPORTANCE: The reprogramming of lipid metabolism is a significant feature of tumors, yet the circulating levels of fatty acids in lung cancer patients remain to be explored. Moreover, the association between fatty acid levels and related factors, including nutritional intake, tumor metabolism, and tumor immunity, has been rarely discussed. OBJECTIVES: To explore the differences in serum free fatty acids between lung cancer patients and healthy controls, and investigate the factors associated with this phenomenon. DESIGN AND PARTICIPANTS: A case-control study enrolled 430 primary lung cancer patients and 430 healthy controls. The whole population had a medium [Q1, Q3] age of 48.0 [37.0, 58.9] years, with females comprising 56% of the participants. The absolute quantification of 27 serum free fatty acids (FFAs) was measured using a liquid chromatography-mass spectrometry (LC-MS/MS) detection. Data, including dietary intake, blood indicators, and gene expression of lung tissues, were obtained from questionnaires, blood tests, and RNA-sequencing. Statistical differences in FFA levels between lung cancer patients and healthy controls were investigated, and related contributing factors were explored. RESULTS: Levels of 22 FFAs were significantly higher in lung cancer patients compared to those in healthy controls, with fold changes ranging from 1.14 to 1.69. Lung cancer diagnosis models built with clinical and FFA features yielded an area under the receiver operating characteristic curve (AUROC) of 0.830 (0.780-0.880). Total fatty acids (TFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) showed no significant dietary-serum associations, indicating that the elevations might not be attributed to an excessive intake of relevant fatty acids from the diet. For RNA-sequencing of lung tissues, among the 68 lipid metabolism genes, 26 genes showed significant upregulation (FDR < 0.05), while 33 genes exhibited significant downregulation, indicating the involvement of the fatty acids in the tumor metabolism. Through joint analysis with immune cells and inflammatory factors in the blood, fatty acids might exert suppressing effects on tumor immunity. CONCLUSIONS: Lung cancer patients had elevated levels of serum free fatty acids compared to healthy individuals. The elevations might not be attributed to an excessive intake of relevant fatty acids from the diet but related to pathological factors of tumor metabolism and immunity. These findings will complement research on fatty acid metabolism of lung cancer and provide insights into potential intervention targets.

Expression and diagnostic value of serum free light chain in lung cancer. / è¡€æ¸ æ¸¸ç¦»è½»é“¾åœ¨è‚ºç™Œä¸­çš„è¡¨è¾¾åŠè¯Šæ–­ä»·å€¼.

OBJECTIVES: The expression of serum free light chain (FLC) is abnormal in various diseases, but its role in lung cancer remains unclear. This study aims to investigate the expression and diagnostic value of serum FLC in lung cancer. METHODS: A total of 80 lung cancer patients treated at Xiangdong Hospital, Hunan Normal University from January to December 2021 were selected as the lung cancer group. Another 80 healthy individuals undergoing routine physical examinations during the same period were chosen as the control group. General information and serum κFLC and λFLC levels were collected for all subjects. Clinical indicators such as serum carcinoembryonic antigen (CEA), cytokeratin fragment antigen 21-1 (CYFRA21-1) levels, tumor diameter, histological type, TNM stage, and lymph node metastasis status were recorded for lung cancer patients. The expression levels of serum FLC [κFLC, λFLC, and FLC (κ+λ)] were compared between the lung cancer group and the control group. Lung cancer patients were grouped based on gender, age, smoking history, tumor diameter, TNM stage, histological type, and lymph node metastasis to compare differences in serum κFLC and λFLC levels. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value of serum FLC alone and in combination with other indicators in lung cancer. RESULTS: The expression levels of serum FLC (κ+λ) and κFLC were significantly higher in the lung cancer group than those in the control group (both P<0.001), while there was no significant difference in serum λFLC levels between the 2 groups (P>0.05). There were no significant differences in serum κFLC levels among lung cancer patients with different tumor diameters, histological types, or TNM stages (all P>0.05); however, serum κFLC levels were higher in lung cancer patients with lymph node metastasis than in those without, with statistical significance (P=0.033). There were no significant differences in serum λFLC levels based on tumor diameter or histological type (both P>0.05), but serum λFLC levels were higher in stage III+IV and lymph node metastatic lung cancer patients compared to stage I+II and non-metastatic patients, with statistical significance (P=0.033 and P=0.019, respectively). The area under the curve (AUC) for κFLC and CEA in diagnosing lung cancer showed no significant difference (P=0.333). The combination of κFLC+CYFRA21-1 had the highest diagnostic efficacy (AUC=0.875) and sensitivity (71.3%). The AUC for the combined diagnosis of κFLC+λFLC+CEA+CYFRA21-1 was 0.915 (95% CI 0.860 to 0.953, P<0.001). CONCLUSIONS: Serum FLC is highly expressed in lung cancer and is associated with its invasion and metastasis. Serum FLC, particularly κFLC, has diagnostic value for lung cancer, and the combined detection of FLC, CEA, and CYFRA21-1 offers the best diagnostic efficacy.

A human-serum-free medium can induce more infectious P. falciparum gametocytes than a conventional human-serum-containing medium.

Malaria remains a global health problem, and the standard membrane feeding assay (SMFA) is a key functional assay for development of new interventions to stop malaria transmission from human to mosquito. For SMFA, media with ~ 10% of human serum has been used for infectious gametocyte cultures, however, there are multiple challenges to obtain a suitable human serum. Here we show a human-serum-free culture medium (HSF), which was a mixture of two stem cell culture media and AlbuMAX, supported infectious gametocyte growth. Moreover, the HSF-induced gametocytes elicited significantly higher numbers of oocysts compared to gametocytes cultured with conventional human serum medium (Conv). While some caution is required when comparing percent transmission reducing activity data generated from HSF-SMFA and Conv-SMFA, the HSF method can facilitate the establishment of gametocyte cultures or SMFA by bypassing the need for human serum. Thus, this study will support future development of P. falciparum transmission-blocking interventions.

Establishment of porcine embryonic stem cells in simplified serum free media and feeder free expansion.

BACKGROUND: The establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium. METHODS: We evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions. RESULTS: The pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions. CONCLUSIONS: These results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies.

Heparan Sulfate-Collagen Surface Multilayers Support Serum-Free Microcarrier Culture of Mesenchymal Stem Cells.

The increasing cost of high-volume cultures and dependence on serum and growth factor supplementation limit the affordability of mesenchymal stromal cell (MSC) therapies. This has spurred interest in developing strategies that support adherent cell expansion while reducing raw material costs. Culture surfaces coated with sulfated glycosaminoglycans (GAGs), specifically heparan sulfate (HS), are an alternative to prolong growth factor retention in cell cultures. Unlike heparin, recombinant HS (rHS) offers strong binding affinity for multiple growth factors and extracellular matrix components, such as collagen I, without undesirable anticoagulant effects or xenobiotic health risks. The potential of rHS as a factor reservoir in MSC cultures remains underexplored. This study investigated the impact of rHS on the growth and anti-inflammatory properties of undifferentiated bone marrow MSCs in both planar and microcarrier-based cultures. It was hypothesized that rHS would enable MSC growth with minimal growth factor supplementation in a sulfation level-dependent manner. Cell culture surfaces were assembled via the layer-by-layer (LbL) method, combining alternating collagen I (COL) and rHS. These bilayers support cell adhesion and enable the incorporation of distinct sulfation levels on the culture surface. Examination of pro-mitogenic FGF and immunostimulatory IFN-γ release dynamics confirmed prolonged availability and sulfate level dependencies. Sulfated surfaces supported cell growth in low serum (2% FBS) and serum-free (SF) media at levels equivalent to standard culture conditions. Cell growth on rHS-coated surfaces in SF was comparable to that on heparin-coated surfaces and commercial surface-coated microcarriers in low serum. These growth benefits were observed in both planar and microcarrier (µCs) cultures. Additionally, rHS surfaces reduced ß-galactosidase expression relative to uncoated surfaces, delaying cell senescence. Multivariate analysis of cytokines in conditioned media indicated that rHS-containing surfaces enhanced cytokine levels relative to uncoated surfaces during IFN-γ stimulation and correlated with decreased pro-inflammatory macrophage activity. Overall, utilizing highly sulfated rHS with COL reduces the need for exogenous growth factors and effectively supports MSC growth and anti-inflammatory potency on planar and microcarrier surfaces under minimal factor supplementation.

[Correlation between Morphological Typing and Monoclonality of Bone Marrow Plasma Cells and Its Diagnostic Value for High-Risk Smoldering Multiple Myeloma].

OBJECTIVE: To investigate the correlation between morphological typing and monoclonality of bone marrow plasma cells, and explore the diagnostic value of plasma cell morphological typing for high-risk smoldering multiple myeloma(HR-SMM). METHODS: The correlation between the morphological characteristics and the monoclonality of bone marrow plasma cells was analyzed in 84 patients with HR-SMM who treated in our hospital. The consistency of morphologically abnormal bone marrow plasma cells with serum free light chain (sFLC) ratio, next-generation sequencing (NGS) detection results, and its correlation with monoclonal plasma cells detected by flow cytometry (FCM) were further verified. The immunoglobulin types and levels of non-involved immunoglobulins in serum of the patients were detected, and the distribution of plasma cell clusters in patients with different disease was observed. RESULTS: The mean percentage of mature plasma cells were decreased successively in the order of reactive plasmacytosis (RP) group, monoclonal gammopathy of undetermined significance (MGUS) group, smoldering multiple myeloma (SMM) group, HR-SMM group and multiple myeloma (MM) group; while the mean percentage of immature, primitive, reticular and flaming plasma cells were increased successively in the order of RP group, MGUS group, SMM group, and HR-SMM group, and the difference between any two groups was statistically significant (P < 0.05).The average proportion of abnormal plasma cells in the bone marrow of HR-SMM patients was 96.2% of the total plasma cells. The proportion of abnormal plasma cells were in good agreement with the sFLC ratio and the results of NGS detection in HR-SMM patients (kappa=0.879 and kappa=0.891, both >0.75)，and showed good correlation with the monoclonal plasma cells with immunophenotype of CD45-/CD38+/CD138+/CD56+/CD19-( γ=0.825). The levels of non-involved immunoglobulin in IgG, IgA and IgM type HR-SMM patients were all decreased by more than 25% compared with the normal reference range, and the differences were statistically significant (P < 0.05). There was no significant difference in the distribution ratio of plasma cell clusters among different disease groups (P >0.05). CONCLUSION: In HR-SMM patients, the immature, primitive, reticular and flaming plasma cells in bone marrow are considered as abnormal plasma cells, and they are correlated with monoclonal plasma cells. The proportion of abnormal plasma cells in total plasma cells of bone marrow and the reduction extent of non-involved immunoglobulin level in patients have certain reference value for the diagnosis of HR-SMM.

Distinctive small molecules blend: Promotes lacrimal gland epithelial cell proliferation in vitro and accelerates lacrimal gland injury repair in vivo.

PURPOSE: This study aims to develop a novel serum-free culture strategy containing only two small molecules, Y27632 and SB431542 (2C), for in vitro expansion of mouse lacrimal gland epithelial cells (LGECs) and investigate an innovative therapeutic approach for lacrimal gland (LG) injury. METHODS: LGECs proliferative capacity was assessed by cell counting, crystal violet staining, qRT-PCR and immunofluorescence. Cell differentiation was achieved by manipulating culture conditions and assessed by qRT-PCR and AQP5 immunofluorescence. LGECs were seeded in Matrigel for three-dimensional culture and assessed by qRT-PCR and immunofluorescence. Secretory function of the cultures was assayed by ELISA. In vivo, 2C injection verified its reparative capacity in a mouse LG injury model. Corneal fluorescein staining, phenol red cotton thread, H&E, immunofluorescence and Western blot were used to assess LG injury repair. RESULTS: LGECs cultured with 2C exhibited high expression of stemness/proliferation markers and maintained morphology and proliferative capacity even after the tenth passage. Removal of 2C was efficacious in achieving LGECs differentiation, characterized by the increased AQP5 expression and LTF secretion. 3D spheroids cultured with 2C demonstrated differentiation potential, forming microglandular structures containing multiple LG cell types with secretory functions after 2C removal. In vivo, 2C improved the structural integrity and function of the injured LG. CONCLUSIONS: We present a small molecule combination, 2C, that promotes LGECs expansion and differentiation in vitro and accelerates LG injury repair in vivo. This approach has potential applications for providing a stable source of seed cells for tissue engineering applications, providing new sights for LG-related diseases treatment.

Development of scaffold-free tissue-engineered constructs derived from mesenchymal stem cells with serum-free media for cartilage repair and long-term preservation.

Synovial mesenchymal stem cells (sMSCs) have great potential for cartilage repair, but their therapeutic design to avoid adverse effects associated with unknown factors remains a challenge. In addition, because long-term preservation is indispensable to maintain high quality levels until implantation, it is necessary to reduce their fluctuations. This study aimed to investigate the properties and feasibility of novel scaffold-free tissue-engineered constructs using serum-free media and to develop long-term preservation methods. sMSCs were cultured in serum-free media, seeded at high density in a monolayer, and finally developed as a sheet-like construct called "gMSC1". The properties of frozen gMSC1 (Fro-gMSC1) were compared with those of refrigerated gMSC1 (Ref-gMSC1) and then examined by their profile. Chondrogenic differentiation potential was analyzed by quantitative real-time polymerase chain reaction and quantification of glycosaminoglycan content. Xenografts into the cartilage defect model in rats were evaluated by histological staining. gMSC1 showed nearly similar properties independent of the preservation conditions. The animal experiment demonstrated that the defect could be filled with cartilage-like tissue with good integration to the adjacent tissue, suggesting that gMSC1 was formed and replaced the cartilage. Furthermore, several chondrogenesis-related factors were significantly secreted inside and outside gMSC1. Morphological analysis of Fro-gMSC1 revealed comparable quality levels to those of fresh gMSC1. Thus, if cryopreserved, gMSC1, with no complicated materials or processes, could have sustained cartilage repair capacity. gMSC1 is a prominent candidate in novel clinical practice for cartilage repair, allowing for large quantities to be manufactured at one time and preserved for a long term by freezing. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-024-00637-y.

Spheroid trilineage differentiation model of primary mesenchymal stem/stromal cells under hypoxia and serum-free culture conditions.

Due to their unique properties, human mesenchymal stem/stromal cells (MSCs) possess tremendous potential in regenerative medicine, particularly in cell-based therapies where the multipotency and immunomodulatory characteristics of MSCs can be leveraged to address a variety of disease states. Although MSC-based cell therapeutics have emerged as one of the most promising medical treatments, the clinical translation is hampered by the variability of MSC-based cellular products caused by tissue source-specific differences and the lack of physiological cell culture approaches that closely mimic the human cellular microenvironment. In this study, a model for trilineage differentiation of primary adipose-, bone marrow-, and umbilical cord-derived MSCs into adipocytes, chondrocytes and osteoblasts was established and characterized. Differentiation was performed in spheroid culture, using hypoxic conditions and serum-free and antibiotics-free medium. This platform was characterized for spheroid diameter and trilineage differentiation capacity reflecting functionality of differentiated cells, as indicated by lineage-specific extracellular matrix (ECM) accumulation and expression of distinct secreted markers. The presented model shows spheroid growth during the course of differentiation and successfully supports trilineage differentiation for MSCs from almost all tissue sources except for osteogenesis of umbilical cord-derived MSCs. These findings indicate that this platform provides a suitable and favorable environment for trilineage differentiation of MSCs from various tissue sources. Therefore, it poses a promising model to generate highly relevant biological data urgently required for clinical translation and therefore might be used in the future to generate in vitro microtissues, building blocks for tissue engineering or as disease models.

Expansion of human allogeneic liver-derived progenitor cells for liver regenerative therapy in serum-free culture conditions.

Human allogeneic liver-derived progenitor cells (HALPCs) display advanced ability to differentiate into hepatocyte-like cells and exhibit potent immunomodulatory, anti-inflammatory, and anti-fibrotic properties. HALPCs have been successfully manufactured under good manufacturing practice (GMP) and are currently in clinical development. A previous phase 2a trial demonstrated the safety of peripheral intravenous infusions of HALPCs and preliminary evidence of the cells' properties to restore liver function in patients with acute-on-chronic liver failure (ACLF), thus potentially improving their survival. A phase 2b trial is currently ongoing across multiple centers (NCT04229901) to obtain proof-of-concept on efficacy and additional safety. HALPCs are currently manufactured using fetal bovine serum (FBS), which can reveal qualitative and quantitative variations between batches. The use of serum-free medium (SFM) represents an alternative means to overcome this variability while also complying fully with regulations. The aim of this study was to compare current FBS-containing culture conditions with two industry-available GMP-compliant SFMs: StemMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and PRIME-XV (FUJIFILM Irvine Scientific, Santa Ana, California, USA). The proliferation of HALPCs was significantly stimulated by both SFMs, which shortened both their emergence period and population doubling time. This effect was correlated with a significant improvement in their genetic stability as analyzed by conventional karyotyping. The expression profile (identity and purity) and functionality of HALPCs cultured in SFM were maintained, as demonstrated by flow cytometry and enzyme-linked immunoassay (ELISA), respectively. Their potency, evaluated via prostaglandin E2 (PGE2) secretion, showed a similar effect on CD4+ T-cell proliferation in FBS and SFM conditions. Furthermore, a greater proportion of HALPCs cultured in SFM showed enhanced expression of tissue factor (CD142) compared with the FBS condition. Altogether, SFM conditions enabled consistent HALPC quality to be achieved without altering their expression and functional profiles.

Advances in serum-free media for CHO cells: From traditional serum substitutes to microbial-derived substances.

The Chinese hamster ovary (CHO) cell is an epithelial-like cell that produces proteins with post-translational modifications similar to human glycosylation. It is widely used in the production of recombinant therapeutic proteins and monoclonal antibodies. Culturing CHO cells typically requires the addition of a certain proportion of fetal bovine serum (FBS) to maintain cell proliferation and passaging. However, serum is characterized by its complex composition, batch-to-batch variability, high cost, and potential risk of exogenous contaminants such as mycoplasma and viruses, which impact the purity and safety of the synthesized proteins. Therefore, search for serum alternatives and development of serum-free media for CHO-based protein biomanufacturing are of great significance. This review systematically summarizes the application advantages of CHO cells and strategies for high-density expression. It highlights the developmental trends of serum substitutes from human platelet lysates to animal-free extracts and microbial-derived substances and elucidates the mechanisms by which these substitutes enhance CHO cell culture performance and recombinant protein production, aiming to provide theoretical guidance for exploring novel serum alternatives and developing serum-free media for CHO cells.

Targeted metabolomic analysis of serum free fatty acids: Lipidomics disturbance in patients with superior limbic keratoconjunctivitis.

This study aimed to identify and quantify free fatty acids (FFAs), secretory phospholipase A2 group IIa (sPLA2-IIa) and cytosolic phospholipase A2 (cPLA2) in serum of superior limbic keratoconjunctivitis (SLK) patients and explored the association between FFAs, sPLA2-IIa and cPLA2 variations and SLK. Targeted metabolomic analysis of FFAs in serum was performed by gas chromatography tandem mass spectrometry (GC-MS/MS) analysis on 16 SLK patients (43.88 ± 7.88 years; female: 62.50%) and 25 healthy controls (43.12 ± 7.88 years; female: 64.00%). Qualitative and absolute quantitative results of FFAs were obtained and classified according to gender and thyroid tests. Differential lipid metabolites, metabolomic pathways and biomarkers were further evaluated. The serum sPLA2-IIa and cPLA2 were determined by enzyme linked immunosorbent assay (ELISA). Among 40 FFAs identified, 6 FFAs showed significant changes (P < 0.05) in SLK patients, including 4 decreased and 2 increased. They were mainly related to unsaturated fatty acid biosynthesis, α-linolenic acid and linoleic acid metabolism, and fatty acid biosynthesis. When dividing the data by gender or abnormal thyroid tests, some comparable FFAs alterations displayed in SLK patients. The ROC analysis revealed that the AUC values of linoleic acid, γ-linolenic acid, cis-8,11,14-eicosatrienoic acid, stearic acid, and palmitic acid, were all greater than 0.8. The serum concentrations of sPLA2-IIa and cPLA2 in patients with SLK were significantly higher than that in healthy controls. Lipidomics disturbance might be the potential mechanism of SLK. Serum FFA biomarkers associated with SLK have potential for the diagnosis and treatment of the disease.

Expansion and Storage of Insect Cells and Virus Stocks.

Healthy insect cell cultures are critical for any method described in this book, including making productive baculovirus banks, protein or AAV expression, and determining viral titers. This chapter describes cell maintenance in shake flasks using serum-free conditions and the expansion of virus stocks from a single plaque purified virus. Insect cells can be passaged over multiple generations, but as the cells may undergo changes over multiple passages, limiting the use of your cells to a defined number of passages such as 50 passages is recommendable. Baculovirus stocks once created using serum-free media are not very stable at 4-8 °C. This chapter also includes a simple method to store cells from an early cell passage and your virus stock in liquid nitrogen.

[The Value of sFLC and Serum Calcium in the Diagnosis and Prognosis of Multiple Myeloma Patients].

OBJECTIVE: To investigate the value of serum free light chain (sFLC) and serum calcium ion in the diagnosis and prognosis of multiple myeloma (MM). METHODS: Forty patients with MM treated in Henan Provincial People's Hospital from January 2018 to January 2022 were selected as the observation group, and 40 healthy volunteers were selected as the control group. The differences of sFLC-κ、sFLC-λ、sFLC-κ/λ, serum calcium ions, etc between the two groups were compared. Meanwhile, the differences of sFLC-κ、sFLC-λ、sFLC-κ/λ, serum calcium ions, etc in different international staging systems (ISS), chemotherapy efficacy and prognosis patients were analyzed. RESULTS: The levels of sFLC-κï¼»(98.39±21.19) vs (12.01±4.45) mg/Lï¼½, sFLC-λï¼»(210.20±45.54) vs (14.10±5.11) mg/Lï¼½ and proportions of hypocalcemia （65% vs 0） in the observation group were significantly higher than those in the control group (P < 0.05), while sFLC-κ/ λ ratio［（0.44±0.10） vs (0.87±0.12)ï¼½ and serum calcium ions ［（1.98±0.46） vs （2.42±0.40）mmol/Lï¼½ were significantly lower than those in the control group (P < 0.05). The sFLC-κ, sFLC-λ, the proportion of hypocalcemia and the course of hypocalcemia in ISS stage III patients in the observation group were significantly higher than those in stage I and II patients (P < 0.05), while sFLC-κ/λ ratio, and serum calcium ions were significantly lower than those in stage I and II patients (P < 0.05). The levels of sFLC-κ ［（107.76±21.22） vs （94.67±20.11）mg/Lï¼½, sFLC- λ［（245.54±41.12） vs （205.54±50.22）mg/Lï¼½ of patients with hypocalcemia in the observation group was significantly higher than those without hypocalcemia (P < 0.05), while the sFLC-κ/λ ratio was significantly lower than those without hypocalcemia ［（0.42±0.04） vs （0.47±0.06）；P < 0.05ï¼½. The levels of sFLC-κ ï¼»(107.29±20.14） vs （ 91.11±18.92）mg/Lï¼½, sFLC-λ［（247.98±42.26） vs （179.29±39.32）mg/Lï¼½ in patients with ineffective chemotherapy were significantly higher than those in patients with effective chemotherapy (P < 0.05), while the sFLC-κ/λ ratio was significantly lower than those in patients with effective chemotherapy ï¼»(0.43±0.10) vs (0.50±0.09)；P < 0.05）］. The area under the ROC curve for sFLC-κ, sFLC-λ, sFLC-κ/λ predicting ineffective chemotherapy was 0.803, 0.793 and 0.699 respectively, P < 0.05. There was no significant difference in sFLC-κ, sFLC-λ, sFLC-κ/λ ratio, serum calcium ion, hypocalcemia ratio and hypocalcemia course between survival and death patients (P >0.05). CONCLUSION: sFLC and serum calcium are related to ISS stage of MM patients. sFLC level has a certain value to predict the curative effect of chemotherapy in MM patients. However, the prognostic values of sFLC and serum calcium are not yet confirmed for MM patients.

Recombinant Protein Production from Stable CHO Cell Pools.

The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or functional studies as well as for their characterization as biotherapeutics. While transient gene expression (TGE) in mammalian cells constitutes a rapid and well-established approach, non-clonal stably transfected cells, or "pools," represent another option, which is especially attractive when recurring productions of the same protein are required. From a culture volume of just a few liters, stable pools can provide hundreds of milligrams to gram quantities of high-quality secreted recombinant proteins.In this chapter, we describe a highly efficient and cost-effective procedure for the generation of Chinese Hamster Ovary cell stable pools expressing secreted recombinant proteins using commercially available serum-free media and polyethylenimine (PEI) as the transfection reagent. As a specific example of how this protocol can be applied, the production and downstream purification of recombinant His-tagged trimeric SARS-CoV-2 spike protein ectodomain (SmT1) are described.