High-density Cultures Research Articles

Effect of backwash as a strategy for biofouling control in the submerged ceramic membrane bioreactor for high-density cultivations: Process optimization and fouling mechanism at pilot scale

Submerged membrane bioreactors are effective tools for synergistic production of valuable compounds, cell retention in the bioreactor, and separation of products from the reaction solution. However, membrane fouling is a problem that impairs efficiency of processes involving submerged membrane bioreactors. One of the mechanical methods for efficient fouling mitigation is backwash. Therefore, this study investigated backwash as a method for mitigating biofouling in a submerged ceramic membrane bioreactor, specifically in terms of separation of Saccharomyces cerevisiae from the suspension. To identify the key factors affecting the dynamic flux and their interactions, a Fractional Factorial Design (FFD) was employed based on the key operating variables (2 levels, 5 variables). The average flux model showed a dependency on the flow rate of backwash, time between backwash cycles, backwash duration, cell concentration, and separation process time (adjusted R2 = 0.99). Furthermore, the effect of backwash on flux recovery was assessed in comparison to separation processes without backwash. It was found that after the application of backwash, up to 100 % of the initial flux could be recovered. In contrast, only around 20 % of the initial flux could be maintained after the process without backwash. The optimized conditions of backwash were found to be a flow rate of backwash at 1500 mL min−1, time between backwash cycles 5 min, backwash duration 5 sec, cell concentration 20 g dry weight per liter, and separation process time 0.5 h. The mechanism of membrane fouling was determined to be the deposition of yeast cells on the membrane surface and the blocking of pores inside the membrane by sorbitan monostearate as a reagent present in the yeast suspension.

Effects of Lactobacillus plantarum and Bacillus subtilis on growth, immunity and intestinal flora of largemouth bass(Micropterus salmoides)

Probiotics play a crucial role in largemouth bass aquaculture, primarily in enhancing water quality, preventing fish diseases, promoting largemouth bass growth, and boosting immunity in high-density culture conditions. The experiment was actualized by adding Lactobacillus plantarum, Bacillus subtilis and a mixture of probiotic (L. plantarum and B. subtilis 1:1) in the culture water of largemouth bass for 42 days, respectively, to explore the effect of the above two probiotics on the growth performance and immunity ability of largemouth bass (Micropterus salmoides).Results: At the level of phylum, the relative abundance of enteric flora in Tenericutes of largemouth bass in L. plantarum added group was significantly higher than that in control group (p < 0.05). Conversely, the relative abundance of Proteobacteria was significantly lower than that of control group (p < 0.05). Similarly, at the genus level, the relative abundance of Mycoplasma and Leifsonia was significantly higher than that of control group (p < 0.05), while the relative abundance of Aeromonas was significantly lower in the L. plantarum added group compared to the control group (p < 0.05). Compared with the control group, there were no significant differences in phylum level in the B. subtilis added group (p < 0.05), and the relative abundance of Plesiomonas was significantly increased (p < 0.05), the relative abundance of Aeromonas was significantly decreased at genus level (p < 0.05). The relative abundance of Plesiomonas and Parabacteroides in the mixed probiotic treatment group was significantly increased (p < 0.05), the relative abundance of Aeromonas was significantly decreased at the level of genus (p < 0.05). The metabolites of carboxylic acid and its derivatives, organic oxygen compound, glycerophospholipid, lip acyl and other categories in enteric tissues of all treatment groups had significant changes (p < 0.05), enteric bacterial secondary metabolites such as SCFAs and 1-Phosphatidyl-D-myo-inositol were significantly increased (p < 0.05). The results showed that the addition of L. plantarum and B. subtilis in water could promote the growth of largemouth bass and increase its nonspecific immunity by enhancing the stability of enteric flora, increasing efficiency of enteric digestion and absorption and promoting the anti-inflammatory and immune ability of the body through secondary metabolites of enteric microbe.

Impact of the hypoxic microenvironment on spermatogonial stem cells in culture.

The stem cell niche plays a crucial role in the decision to either self-renew or differentiate. Recent observations lead to the hypothesis that O2 supply by blood and local O2 tension could be key components of the testicular niche of spermatogonial stem cells (SSCs). In this study, we investigated the impact of different hypoxic conditions (3.5%, 1%, and 0.1% O2 tension) on murine and human SSCs in culture. We observed a deleterious effect of severe hypoxia (1% O2 and 0.1% O2) on the capacity of murine SSCs to form germ cell clusters when plated at low density. Severe effects on SSCs proliferation occur at an O2 tension ≤1% and hypoxia was shown to induce a slight differentiation bias under 1% and 0.1% O2 conditions. Exposure to hypoxia did not appear to change the mitochondrial mass and the potential of membrane of mitochondria in SSCs, but induced the generation of mitochondrial ROS at 3.5% and 1% O2. In 3.5% O2 conditions, the capacity of SSCs to form colonies was maintained at the level of 21% O2 at low cell density, but it was impossible to amplify and maintain stem cell number in high cell density culture. In addition, we observed that 3.5% hypoxia did not improve the maintenance and propagation of human SSCs. Finally, our data tend to show that the transcription factors HIF-1α and HIF-2α are not involved in the SSCs cell autonomous response to hypoxia.

Microglia-mediated drug substance transfer promotes chemoresistance in brain tumors: insights from an in vitro co-culture model using GCV/Tk prodrug system

BackgroundIt is well known that tumor-associated macrophages (TAMs) play essential roles in brain tumor resistance to chemotherapy. However, the detailed mechanisms of how TAMs are involved in brain tumor resistance are still unclear and lack a suitable analysis model.MethodsA BV2 microglial cells with ALTS1C1 astrocytoma cells in vitro co-culture system was used to mimic the microglia dominating tumor stroma in the tumor invasion microenvironment and explore the interaction between microglia and brain tumor cells.ResultsOur result suggested that microglia could form colonies with glioma cells under high-density culturing conditions and protect glioma cells from apoptosis induced by chemotherapeutic drugs. Moreover, this study demonstrates that microglia could hijack drug substances from the glioma cells and reduce the drug intensity of ALTS1C1 via direct contact. Inhibition of gap junction protein prevented microglial-glioma colony formation and microglia-mediated chemoresistance.ConclusionsThis study provides novel insights into how glioma cells acquire chemoresistance via microglia-mediated drug substance transferring, providing a new option for treating chemo-resistant brain tumors.

Mechanism analysis of methionine inhibiting growth of Bifidobacterium bifidum

Bifidobacterium bifidum rarely utilises yeast extract, which causes significant challenges in its industrial production. This study characterised the utilisation of amino acids by B. bifidum to improve the viability of the strain. In this study, the proliferation effect of B. bifidum in different nitrogen sources was determined, and the amino acid contents of different nitrogen sources were analysed, which indicated that hydrophobic amino acids may inhibit B. bifidum growth. Among different hydrophobic amino acids, methionine was identified as the strongest growth inhibitor. Furthermore, the intracellular pH and NH4+ contents of B. bifidum CCFM16 were higher in methionine-free medium. Among the NH3-producing enzymes related to amino acid metabolism, the metC gene was significantly transcribed in methionine-free medium. In addition, tcyP, metB, metC and metE genes encoding cysteine transporter proteins and the conversion pathway of cysteine to methionine were transcribed 2.03-fold, 7.03-fold, 3.05-fold and 2.92-fold, respectively, in methionine-free medium compared to methionine-containing medium. Compared with methionine-containing medium, the activity of tricarboxylic acid cycle rate-limited enzyme isocitrate dehydrogenase in methionine-free medium also increased from 198.89 to 410.56 nmol/min/mg protein, which probably enhanced the carbon flux and promoted growth. Meanwhile, the activity of pyrroline-5-carboxylate reductase, an enzyme that synthesises proline, increased from 14.76 to 35.28 nmol/min/mg protein, which promoted proline synthesis in response to osmotic stress. Cyclopropane fatty acids were also significantly higher in methionine-free medium, which may promote growth by decreasing cell membrane permeability to protons and enhancing cell membrane fluidity. This study provides a theoretical basis for high-density culture of B. bifidum.

Allelic variation of BBX24 is a dominant determinant controlling red coloration and dwarfism in pear.

Variation in anthocyanin biosynthesis in pear fruit provides genetic germplasm resources for breeding, while dwarfing is an important agronomic trait, which is beneficial to reduce the management costs and allow for the implementation of high-density cultivation. Here, we combined bulked segregant analysis (BSA), quantitative trait loci (QTL), and structural variation (SV) analysis to identify a 14-bp deletion which caused a frame shift mutation and resulted in thepremature translation termination of a B-box (BBX) family of zinc transcription factor, PyBBX24, and its allelic variation termed PyBBX24ΔN14. PyBBX24ΔN14 overexpression promotes anthocyanin biosynthesis in pear, strawberry, Arabidopsis, tobacco, and tomato, while that of PyBBX24 did not. PyBBX24ΔN14 directly activates the transcription of PyUFGT and PyMYB10 through interaction with PyHY5. Moreover, stable overexpression of PyBBX24ΔN14 exhibits a dwarfing phenotype in Arabidopsis, tobacco, and tomato plants. PyBBX24ΔN14 can activate the expression of PyGA2ox8 via directly binding to its promoter, thereby deactivating bioactive GAsand reducing the plant height. However, the nuclear localization signal (NLS) and Valine-Proline (VP) motifs in the C-terminus of PyBBX24 reverse these effects. Interestingly, mutations leading to premature termination of PyBBX24 were also identified in red sports of un-related European pear varieties. We conclude that mutations in PyBBX24 gene link both an increase in pigmentation and a decrease in plantheight.

Silicone rubber membrane devices permit islet culture at high density without adverse effects.

Conventional culture conditions, such as in T-flasks, require that oxygen diffuse through the medium to reach the islets; in turn, islet surface area density is limited by oxygen availability. To culture a typical clinical islet preparation may require more than 20 T-175 flasks at the standard surface area density of 200IE/cm2. To circumvent this logistical constraint, we tested islets cultured on top of silicon gas-permeable (GP) membranes which place islets in close proximity to ambient oxygen. Oxygenation of individual islets under three culture conditions, standard low-density, non-GP high density, and GP high density, were first modeled with finite element simulations. Porcine islets from 30 preparations were cultured for 2days in devices with GP membrane bottoms or in paired cultures under conventional conditions. Islets were seeded at high density (HD, ∼4000IE/cm2, as measured by DNA) in both GP and non-GP devices. In simulations, individual islets under standard culture conditions and high density cultures on GP membranes were both well oxygenated whereas non-GP high density cultured islets were anoxic. Similarly, compared to the non-GP paired controls, islet viability and recovery were significantly increased in HD GP cultures. The diabetes reversal rate in nude diabetic mice was similar for HD GP devices and standard cultures but was minimal with non-GP HD cultures. Culturing islets in GP devices allows for a 20-fold increase of islet surface area density, greatly simplifying the culture process while maintaining islet viability and metabolism.

Tracking Tau in Neurons: How to Transfect and Track Exogenous Tau in Primary Neurons.

Primary murine neurons have proved to be an essential tool for the general investigation of neuronal polarity, polarized Tau distribution, and Tau-based neuronal dysfunction in disease paradigms. However, mature primary neurons are notoriously difficult to transfect with non-viral approaches and are very sensitive to cytoskeletal manipulation and imaging. Furthermore, standard non-viral transfection techniques require the use of a supportive glial monolayer or high-density cultures, both of which interfere with microscopy. Here we provide a simple non-viral liposome-based transfection method that enables transfection of Tau in low levels comparable to endogenous Tau. This allows the investigation of, for example, distribution and trafficking of Tau, without affecting other cytoskeleton-based parameters such as microtubule density or microtubule-based transport. Using this protocol, we achieve a profound transfection efficiency but avoid high overexpression rates. Importantly, this transfection method can be applied to neurons at different ages and is also suitable for very old cultures (up to 18days in vitro). In addition, the protocol can be used in cultures without glial support and at suitable cell densities for microscopy-based single cell analysis. In sum, this protocol has proven a reliable tool suitable for most microscopy-based approaches in our laboratory.

Biotechnological lactic acid production from low-cost renewable sources via anaerobic microbial processes.

Lactic acid (LA) production from microbial fermentation using low-cost renewable sources has emerged as an attractive alternative to the use of petroleum-based products. This approach not only offers sustainable solutions for waste management but also enables the production of value-added products in an eco-friendly manner. However, to make this approach economically viable, optimizing the production process for high yield, productivity, and purity while minimizing costs is crucial. To address these challenges, various approaches have been proposed, including the use of neutralizing agents, high cell density cultures, co-cultures, fed-batch fermentation, and product removal strategies. Overall, this review underscores the potential of microbial fermentation for LA production as a sustainable and cost-effective solution to meet the growing demand for eco-friendly products. Further optimization of fermentation processes and the development of new microbial strains and fermentation techniques are key to advancing this approach. The production of LA through microbial fermentation presents a sustainable and eco-friendly solution to the increasing demand for eco-friendly products. With continued innovation, we can expect to see a significant reduction in the environmental impact of industrial processes, coupled with a more cost-effective and high-purity source of lactic acid for various industries.

A comparative analysis of stem cell differentiation on 2D and 3D substrates using Raman microspectroscopy.

Chondrogenesis is a complex cellular process that involves the transformation of mesenchymal stem cells (MSCs) into chondrocytes, the specialised cells that form cartilage. In recent years, three-dimensional (3D) culture systems have emerged as a promising approach to studying cell behaviour and development in a more physiologically relevant environment compared to traditional two-dimensional (2D) cell culture. The use of these systems provided insights into the molecular mechanisms that regulate chondrogenesis and has the potential to revolutionise the development of new therapies for cartilage repair and regeneration. This study demonstrates the successful application of Raman microspectroscopy (RMS) as a label-free, non-destructive, and sensitive method to monitor the chondrogenic differentiation of bone marrow-derived rat mesenchymal stem cells (rMSCs) in a collagen type I hydrogel, and explores the potential benefits of 3D hydrogels compared to conventional 2D cell culture environments. rMSCs were cultured on 3D substrates for 3 weeks and their differentiation was monitored by measuring the spectral signatures of their subcellular compartments. Additionally, the evolution of high-density micromass cultures was investigated to provide a comprehensive understanding of the process and complex interactions between cells and their surrounding extracellular matrix. For comparison, rMSCs were induced into chondrogenesis in identical medium conditions for 21 days in monolayer culture. Raman spectra showed that rMSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway at a significantly higher rate than the 2D culture cells. 3D cultures expressed stronger and more homogeneous chondrogenesis-associated peaks such as collagens, glycosaminoglycans (GAGs), and aggrecan while manifesting changes in proteins and lipidic content. These results suggest that 3D type I collagen hydrogel substrates are promising for in vitro chondrogenesis studies, and that RMS is a valuable tool for monitoring chondrogenesis in 3D environments.

Inducing Fungal Pelletization Using Affordable Microparticle

Filamentous fungi have been known as one of the potential microorganisms in various industries. One of the isolates with great potential is Mucor irregularis that offer substantial potential to their growth characteristics. In submerged cultures, these microorganisms often aggregate into mycelia, enabling high-density cultivation and enhanced productivity. M irregularis have a high lipid content of 43.46% and a yield of 3.28 g/L. To further lipid-rich biomass production, pelletization is explored to involve the addition of microparticles like magnesium silicate and calcium carbonate. Microparticles have demonstrated the ability to control growth and enhance biomass in various strains. This study investigates the impact of microparticle addition on M. irregularis biomass production and pellet formation. Preliminary tests reveal that the addition of magnesium silicate microparticles (0, 1, 2, and 3 g/L) induces pellet formation, with the 2 g/L treatment yielding optimal results. Microscopic observations confirm that higher magnesium silicate concentrations result in more compact pellets. Biomass production peaks at 72 hours of incubation, reaching 3.09 ± 0.43 g/L, while the largest pellet diameter of 1.27 mm occurs at 48 hours of incubation. This research offers insights into enhancing biomass production and pellet formation in M. irregularis, holding promise for diverse applications.

Fry Counting Method in High-Density Culture Based on Image Enhancement Algorithm and Attention Mechanism

Fry Counting Method in High-Density Culture Based on Image Enhancement Algorithm and Attention Mechanism

Transcriptome analysis revealed regulatory mechanisms of light and culture density on free-living sporangial filaments of Neopyropia yezoensis (Rhodophyta)

Previous research indicated that free-living sporangial filament keep hollow morph under high-culture density and form bipartite cells under low-culture density, while the following conchospore release was inhibited by high light. Here, we further explored the molecular bases of these affects caused by light and culture density using a transcriptome analysis. Many differentially expressed genes (DEGs) related to carbon dioxide concentration and fixation, photosynthesis, chlorophyll synthesis and nitrogen absorption were upregulated under high-light conditions compared with low-light conditions, indicating the molecular basis of rapid vegetative growth under the former. The stress response- and ion transport-related DEGs, as well as the gene encoding the vacuole formation–brefeldin A-inhibited guanine nucleotide exchange protein (BIG, py05721), were highly expressed under high-density conditions, indicating the molecular basis of the hollow morph of free-living sporangial filaments under high-culture density conditions. Additionally, the brefeldin A treatment indicated that the hollow morph was directly influenced by vacuole formation-related vesicle traffic. Others DEGs related to cell wall components, zinc-finger proteins, ASPO1527, cell cycle and cytoskeleton were highly expressed in the low density with low-light group, which might be related to the formation and release of conchospores. These results provide a deeper understanding of sporangial filaments in Neopyropia yezoensis and related species.

Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation.

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

Soybean reduced internode 1 determines internode length and improves grain yield at dense planting

Major cereal crops have benefitted from Green Revolution traits such as shorter and more compact plants that permit high-density planting, but soybean has remained relatively overlooked. To balance ideal soybean yield with plant height under dense planting, shortening of internodes without reducing the number of nodes and pods is desired. Here, we characterized a short-internode soybean mutant, reduced internode 1 (rin1). Partial loss of SUPPRESSOR OF PHYA 105 3a (SPA3a) underlies rin1. RIN1 physically interacts with two homologs of ELONGATED HYPOCOTYL 5 (HY5), STF1 and STF2, to promote their degradation. RIN1 regulates gibberellin metabolism to control internode development through a STF1/STF2–GA2ox7 regulatory module. In field trials, rin1 significantly enhances grain yield under high-density planting conditions comparing to its wild type of elite cultivar. rin1 mutants therefore could serve as valuable resources for improving grain yield under high-density cultivation and in soybean–maize intercropping systems.

Optimization of Growth Medium of Bacillus sp. TTMP20 by Response Surface Methodology

High-density culture is able to obtain high concentration and activity of microbes, which is a key technology for fermentation industry. This will greatly shorten fermentation cycle and reduce production cost. Bacillus sp. TTMP20 is a strain with a high yield of 2,3,5,6-tetramethylpyrazine (TTMP). In this study, the growth medium of Bacillus sp. TTMP20 was optimized. Different carbon/nitrogen sources were investigated, and the best carbon source (glucose) and nitrogen source (yeast extract) was determined. With the concentrations of glucose, yeast extract and pH as influence factors, response surface method was used to optimize the above-mentioned influence factors to maximize cell density of the strain. The optimal growth medium is as follows: 4 g/L of yeast extract, 15.754 g/L of glucose, 2 g/L of K2HPO4, 10 g/L of NaCl, and 6.50 of pH. The predicted and actual maxima are 1.388 and 1.3, respectively. This is 61.4% higher than the cell density of the basic medium. This will provide the adequate seed culture for the subsequent fermentation of TTMP, and improve the application level of Bacillus sp. TTMP20.

CRISPR-Cas9 Mediated Stable Expression of Exogenous Proteins in the CHO Cell Line through Site-Specific Integration

Chinese hamster ovary (CHO) cells are a popular choice in biopharmaceuticals because of their beneficial traits, including high-density suspension culture, safety, and exogenously produced proteins that closely resemble natural proteins. Nevertheless, a decline in the expression of exogenous proteins is noted as culture time progresses. This is a consequence of foreign gene recombination into chromosomes by random integration. The current investigation employs CRISPR-Cas9 technology to integrate foreign genes into a particular chromosomal location for sustained expression. Results demonstrate the successful integration of enhanced green fluorescent protein (EGFP) and human serum albumin (HSA) near base 434814407 on chromosome NC_048595.1 of CHO-K1 cells. Over 60 successive passages, monoclonal cell lines were produced that consistently expressed all relevant external proteins without discernible variation in expression levels. In conclusion, the CHO-K1 cell locus, NC_048595.1, proves an advantageous locus for stable exogenous protein expression. This study provides a viable approach to establishing a CHO cell line capable of enduring reliable exogenous protein expression.

Depth filtration for clarification of intensified lentiviral vector suspension cell culture.

Depth filtration significantly impacts efficiency of lentiviral (LV) vector purification process. However, it is often deprioritized in the overall scope of viral vector manufacturing process optimization. The demand for LV vectors has increased with the rise in disease indications, making it crucial to improve current manufacturing processes. Upstream bioreactor process intensification has enabled cell densities of over 107 viable cells/mL, creating challenges for harvest unit operations. The larger size of LV vectors and their physiochemical similarity to host cell-DNA (HC-DNA) and poor clarification performance causes significant challenges for the subsequent chromatography-based purifications. As a result, a robust and scalable harvest of LV process is needed, especially for LV in vivo therapeutic quality needs. In this study, we systematically evaluated the overlooked yet important issue of depth filtration systems to improve enveloped LV functional vector recovery. We found that an established depth filtration system in process A that provided 94% (n = 6) LV functional recovery could not be translated to intensified Process B cell culture. Hence, the depth filtration process became a bottleneck for the purification performance in an intensified process. We demonstrated an improvement in LV functional vector recovery from 34% to 82% via filter train optimization for an intensified suspension cell culture system (>107 cells/mL with higher titer), while still maintaining a loading throughput of ≥82 L/m2 and turbidity ≤20 NTU. It was demonstrated that the two or three-stage depth filtration scheme is scalable and more suitable for high cell density culture for large scale for LV manufacturing process.

Reversible translocation of acyl-CoA:cholesterol acyltransferase (ACAT) between the endoplasmic reticulum and vesicular structures.

The enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is normally localized in the endoplasmic reticulum (ER) where it can esterify cholesterol for storage in lipid droplets and/or the formation of lipoproteins. Here, we report that ACAT can translocate from the ER into vesicular structures in response to different ACAT inhibitors. The translocation was fast (within minutes), reversible and occurred in different cell types. Interestingly, oleic acid was able to fasten the re-translocation from vesicles back into the reticular ER network. The process of ACAT translocation could also be induced by cyclodextrins, cholesterol, lanosterol (but not 4-cholestene-3 one), 25-hydroxycholesterol, and by certain stress stimuli such as hyperosmolarity (sucrose treatment), temperature change, or high-density cultivation. In vitro esterification showed that ACAT remains fully active after it has been translocated to vesicles in response to hyperosmotic sucrose treatment of the cells. The translocation process was not accompanied by changes in the electrophoretic mobility of ACAT, even after chemical crosslinking. Interestingly, the protein synthesis inhibitor cycloheximide showed a stimulating effect on ACAT activity and prevented the translocation of ACAT from the ER into vesicles.

Physical and Biological Characterizations of Injectable Thermosensitive Poly(N-Isopropylacrylamide)-Collagen I Hydrogels Intended for Meniscus Repair

There has been an increase in the incidence of meniscus injuries worldwide, as well as growing needs for injectable hydrogels with the potentials to promote in situ meniscal tissue repair. This work developed poly(N-isopropylacrylamide)-collagen I composite hydrogels, with a series of different collagen I contents. The hydrogels were injectable under room temperature and started to solidify around 32°C due to their thermosensitivities, meeting the requirements of the intended applications. The addition of collagen I to the poly(N-isopropylacrylamide) provided physical reinforcement to the hydrogels. Rheological characterizations showed that, under room temperature, the complex viscosity, the storage modulus, and the loss modulus, respectively, increased from 0.71 ± 0.35 Pa•s to 110.95 ± 23.22 Pa•s, from 3.28 ± 2.02 Pa to 658.06 ± 128.07 Pa, and from 3.30 ± 1.01 Pa to 308.78 ± 56.67 Pa, as the collagen I content in the hydrogel increased from 0% to 1%. Cell proliferation tests and high-density culture also revealed that collagen I promoted bioactivity of the hydrogels and induced fibrochondrocyte phenotype of the chondrocytes. The expressions of collagen I protein and collagen I a1 gene in the PNIPAm-0.5Coll group were 3.50-fold and 3.64-fold of those in the PNIPAm group. In contrast, the expressions of collagen II protein and collagen II a1 gene were less prominent (respectively, 2.36-fold and 3.34-fold in the PNIPAm-0.5Coll group compared with the PNIPAm group). This phenotyping was believed to be conducive to the regeneration of meniscal tissues. The findings of this study have provided an important basis for future in vivo studies and clinical applications of this type of composite hydrogels.