Designing lysate-based hydrogels for on-demand therapy

Abstract

Hydrogels are three-dimensional hydrophilic networks formed via physical or chemical crosslinking. Owing to their structural mimicry of native extracellular matrices and tunable capacity for therapeutic cargo delivery, they have garnered substantial attention in biomedical engineering. Traditional hydrogels primarily function as passive scaffolds for drug encapsulation; however, recent advances have shifted toward integrating bioactive lysates that extract derived from cells or tissues are rich in growth factors, cytokines, and antigenic components to construct dynamic, multifunctional platforms. Lysate-based hydrogels combine the intrinsic bioactivity of lysate-derived factors (e.g., tumor-specific neoantigens or bacteria-derived pathogen-associated molecular patterns) with the spatiotemporal control afforded by hydrogel matrices. This synergy enables precise modulation of the tumor microenvironment, immune priming, and tissue regeneration. In this review, we highlight the emergence of lysate-based hydrogels, which hold significant guiding value for the entire field of bioengineering. This technology represents an innovation in both design concepts and therapeutic strategies, with potential applications across multiple related disciplines. By proposing strategies to exploit underexplored lysate sources (e.g., microbiota-derived components) and integrate stimuli-responsive materials, this work aims to advance lysate-based hydrogels as next-generation platforms for precision oncology, while balancing biological complexity with engineering reproducibility. Key challenges in the development of lysate-based hydrogels include determining the optimal dosage and composition of lysate-derived factors, as well as coordinating their interactions. The presence of multiple factors endows these hydrogels with pluripotent therapeutic effects, but potential crosstalk between factors may limit their efficacy. This area requires further in-depth exploration in future research.

Graphical abstract

Highlights

• •Lysate hydrogels advance biomedicine by merging biological complexity with engineering precision for future therapies.
• •The diverse bioactive factors in lysates drive therapies across immunotherapy, tissue regeneration, and oncology.
• •Stable, biocompatible lysate hydrogels transform regenerative medicine, drug delivery, and immunoengineering.

1. Introduction

Hydrogels, three-dimensional hydrophilic networks formed through physical or chemical crosslinking, have emerged as pivotal biomaterials in diverse fields of biomedical engineering [[1], [2], [3]]. Conventional hydrogels, however, predominantly rely on synthetic or highly purified natural polymers (e.g., polyvinyl alcohol, chitosan, and polyethylene glycol) for encapsulating single-agent drugs or bioactive molecules. More critically, ultra-pure compounds and high manufacturing expenses restrict the clinical scalability of these hydrogels, hindering the translation of numerous research findings into practical applications. Additionally, the single-component nature of therapeutic agents in traditional hydrogels confers only unidirectional therapeutic effects, failing to address the multifactorial pathogenesis of complex diseases such as chronic wounds and cancer. Further limitations of traditional materials include poor biocompatibility (e.g., polyethylene glycol hydrogels, which generate reactive oxygen species (ROS) that induce protein carbonylation and compromise drug activity) and batch-to-batch variability (e.g., matrigel, where significant differences in the concentration of key proteins between batches directly impair reproducibility). For instance, Anjali et al. [4] investigated a ferulic acid‑loaded self‑cross‑linking hydrogel (ADAGFA), which exhibited excellent physical properties, antibacterial activity, and the ability to promote cell migration and collagen deposition. The study concluded that this hydrogel is highly suitable for treating moderately exudating wounds.

In the history of medical therapy, the conventional paradigm for treating diseases typically involves first extracting or synthesizing active ingredients, which are often derived from metabolites or secretions of cells, tissues, or bacteria. For example, targeted intervention against a single gene or direct silencing of a key gene poses significant technical challenges. Moreover, ensuring the biological safety of drugs, avoiding toxicity caused by high drug concentrations, maintaining the stability of secreted cellular components, and reducing the complexity of target exploration are longstanding issues in the medical community, which fundamentally impede the advancement of therapeutic strategies. In recent years, lysate-based hydrogels have emerged as a transformative alternative, leveraging the intrinsic bioactivity of cellular or tissue lysates to overcome these constraints. A lysate-based hydrogel is defined as an emerging class of hydrogel in which cell, tissue, or bacterial lysates directly participate in hydrogel synthesis and exert primary functional roles. Unlike conventional systems, lysate-based hydrogels harness the natural complexity of biological extracts (e.g., platelet-derived growth factors, tumor-specific antigens, and bacteria-derived pathogen-associated molecular patterns) to achieve multimodal therapeutic effects. For example, lysates can deliver a cocktail of cytokines (e.g., vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)) and proteases that synergistically enhance angiogenesis, tissue remodeling, and immune modulation [[5], [6], [7]]; meanwhile, tumor lysates can prime adaptive immunity through the presentation of neoantigens [8]. The preparation of lysate-based hydrogels prioritizes simplicity and cost-effectiveness, circumventing the laborious purification steps required for synthetic systems. Bioactive components can be efficiently extracted via freeze-thaw cycles or solvent-based lysis protocols and then crosslinked into hydrogels using polymers such as alginate or chitosan. This streamlined process not only reduces production costs but also preserves the native bioactivity of labile proteins, addressing a critical limitation of traditional hydrogels.

The therapeutic versatility of lysate-based hydrogels is further enhanced by their adaptability to diverse biological sources. For example, adipose tissue lysates are rich in collagen and fibroblast growth factor-2 (FGF-2) that have demonstrated promising potential in soft tissue regeneration [9]; osteocyte-derived lysates can regulate bone remodeling by activating osteoblasts and suppressing osteoclasts [10]; and bacterial lysates, which have long been used in vaccines (e.g., Bacillus Calmette-Guérin (BCG)), are now being repurposed to stimulate antitumor immunity via pathogen-associated molecular patterns [11]. Nevertheless, challenges such as optimizing lysis efficiency, standardizing crosslinking protocols, and evaluating long-term biocompatibility require systematic investigation.

The significance of lysate research lies in its ability to drive innovation in therapeutic paradigms and facilitate the validation of lysate-based therapies for additional diseases, as well as the exploration of new lysate sources. The advent of lysate-based therapy has transcended the conventional medical paradigm by simultaneously targeting multiple pathways through a diverse array of factors, offering unique advantages over single purified drugs while ensuring efficacy. Furthermore, the rich repertoire of biological factors in lysates provides a foundation for validating therapeutic effects across a broader spectrum of diseases. Currently, lysate research primarily focuses on platelet and tumor lysates, with limited studies exploring other cell and tissue types. Expanding the scope of lysate sources (e.g., stem cells) represents a promising avenue for extending the therapeutic applications of lysates.

This review synthesizes the current state of lysate-based hydrogel research, focusing on three core aspects: lysate sources, preparation methods, and practical applications. Furthermore, the comparison with conventional pure-component hydrogels reveals several innovative strategies, including the integration of platelet lysates with matrix metalloproteinase-13 (MMP-13) inhibitors for cartilage repair [12] and the combination of tumor lysates with Toll-like receptor 3 (TLR3) agonists for enhanced immunotherapy [13]. Therefore, we underscore their potential to redefine biomaterial design. We further identify underexplored opportunities, including the utilization of plant-derived lysates to innovate biomaterial design, with the aim of guiding future research toward the development of scalable, multifunctional therapeutic platforms.

2. Lysates from different sources

Lysate-based hydrogels represent a rapidly evolving research area. Current studies primarily focus on three categories of lysate-based hydrogels, classified according to their biological sources: platelet lysate-based hydrogels, tumor lysate-based hydrogels, and other specialized lysate-derived hydrogels (Fig. 1).

Fig. 1.

Six primary lysate sources: platelets, tumors, adipose tissue, osteocytes, macrophages, and bacteria. FGF-2: fibroblast growth factor 2; IFN-γ: interferon-γ; IL-1: interleukin-1; IL-12: interleukin-12.

2.1. Platelet lysates

Platelet lysates are cost-effective, multifunctional biological materials enriched with a diverse array of bioactive factors. Their composition includes cytokines (e.g., granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-1), IL-7, IL-8, and interferon-γ (IFN-γ)), chemokines (e.g., C-C motif chemokine ligand 5 (CCL5/RANTES), C-X-C motif chemokine ligand 1–3 (CXCL1–3)), transport proteins (e.g., transferrin, haptoglobin), protease inhibitors (e.g., α1-antitrypsin), growth factors (e.g., insulin-like growth factor-1 (IGF-1), PDGF-AA/AB, transforming growth factor-β1/β2 (TGF-β1/β2), VEGF, epidermal growth factor (EGF), FGF-2), and plasma-derived proteins (e.g., albumin, immunoglobulins) [14,15] (Fig. 2).

Fig. 2.

Composition of bioactive factors in platelet lysates: cytokines, chemokines, transport proteins, protease inhibitors, and growth factors. PDGF-AA/AB: platelet-derived growth factor-AA/AB; IGF-1: insulin-like growth factor-1; VEGF: vascular endothelial growth factor; TGF-β1/β2: transforming growth factor-β1/β2; G-CSF: granulocyte colony-stimulating factor; IL-1: interleukin-1; IL-7: interleukin-7; IL-8: interleukin-8; CCL5: C-C motif chemokine ligand 5; CXCL1: C-X-C motif chemokine ligand 1; CXCL2: C-X-C motif chemokine ligand 2; CXCL3: C-X-C motif chemokine ligand 3.

TGF-β is a key regulator of cell survival, proliferation, differentiation, and tissue homeostasis [16,17]. Among growth factors, brain-derived neurotrophic factor (BDNF) has attracted significant attention for its neuroprotective role in neuronal development and its therapeutic potential in neurological disorders such as Alzheimer's disease [[18], [19], [20], [21]]. Similarly, IGF-1 acts as a hepatokine that modulates metabolic processes. It functions by enhancing insulin sensitivity, glucose uptake, and lipid metabolism, while simultaneously balancing growth hormone and insulin signaling [[22], [23], [24], [25]]. VEGF, particularly isoforms VEGF-C and VEGF-D, drives lymphatic angiogenesis, whereas VEGF-A promotes vascular permeability and blood vessel formation [26,27]. EGF, widely used in clinical wound management, accelerates healing by regulating skin cell activity, resolving inflammation, and restoring the skin barrier [[28], [29], [30], [31], [32]].

The direct therapeutic effect of platelet lysates lies in their ability to rapidly replenish biological factors, which collectively cover multiple physiological processes. For example, platelet aggregation is a critical initial step in wound healing, and platelet lysates provide essential factors for all four stages of wound repair: (1) Coagulation phase: PDGF plays a pivotal role in vasoconstriction and coagulation, facilitating hemostasis and the formation of a temporary extracellular matrix; (2) Inflammatory phase: Inflammatory cytokines such as IL-1 dominate, mediate immune cell recruitment and activation; (3) Proliferative phase: Fibroblasts and endothelial cells are activated by every factors. VEGF promotes angiogenesis, FGF-2 stimulates fibroblast proliferation, TGF-β regulates collagen synthesis, and EGF drives re-epithelialization; (4) Remodeling phase: TGF-β continues to modulate collagen metabolism, while IGF-1 contributes to tissue maturation. Notably, some factors (e.g., PDGF, and TGF-β) exhibit stage-specific functions, highlighting the complexity of their regulatory roles. Timely supplementation of these factors via platelet lysates compensates for their consumption during wound healing, thereby accelerating the repair process. Collectively, the diverse bioactive composition of platelet lysates underscores their versatility in biomedical applications, particularly in regenerative medicine.

Four primary methods are used for platelet lysate preparation: freeze-thaw cycles, solvent/detergent inactivation, ultrasonication, and calcium chloride treatment. The freeze-thaw method is cost-effective and straightforward, typically requiring 3−5 cycles; however, this process may compromise the retention of bioactive factors. Studies have shown that freezing at −196 °C (liquid nitrogen) and thawing at −4 °C better preserves growth factors [33], whereas cycles of −80 °C freezing and 37 °C thawing result in significant degradation of these factors [34]. Despite this, most current studies employ −80 °C/37 °C cycles, as this protocol is more accessible and 37 °C more closely mimics the physiological environment of platelets. The impact of temperature cycles on the therapeutic properties of lysate-based hydrogels remains an interesting topic for further investigation. Solvent/detergent inactivation commonly uses a combination of 1% tri-n-butyl phosphate and 1% Triton X-45 [35]. Ultrasonication, optimized at 20 kHz for 30 min, effectively maintains the integrity of growth factors [36]. To prevent coagulation, heparin is frequently added to lysates; however, its potential adverse effects on patient tissues require careful evaluation [37].

2.2. Tumor lysates

Research on tumor lysates has primarily focused on their application in tumor vaccine development (Fig. 3). Tumor immunotherapy, first clinically explored in 1893 [38], has evolved to incorporate modern strategies such as immune checkpoint inhibitors and T-cell-mediated therapy for solid and hematological malignancies [39]. Among these approaches, tumor vaccines remain critical for cancer prevention and therapeutic intervention.

Fig. 3.

The advantages of fabricating hydrogels from tumor lysates. Autologously resected tumors are rich in antigens (especially neoantigens); tumor lysates are fabricated into hydrogels for tumor therapy.

The immune system targets cancer cells by recognizing tumor-specific antigens (TSAs) or neoantigens [40]. However, the high heterogeneity and complexity of tumors often result in patient-specific neoantigens, complicating the design of broad-spectrum vaccines [41,42]. To address this challenge, tumor cell or tissue lysates (autologous or allogeneic) are increasingly used as antigen reservoirs. Water-soluble components extracted from lysates can be directly incorporated into vaccines [43,44], and lysed tumor cells retain native antigenicity without compromising immune functionality.

Current tumor lysate vaccines are primarily categorized into two formulations: (1) Vaccines derived from water-soluble lysate fractions; (2) Vaccines incorporating whole tumor cell/tissue lysates [45]. Autologous tumor lysates, which contain patient-specific mutations and neoantigens, offer personalized therapeutic relevance that cannot be matched by allogeneic sources [46].

The preparation of tumor cell lysates involves inducing apoptosis or necrosis via four primary methods: freeze-thaw cycles, ultraviolet (UV) radiation, hypochlorous acid (HOCl) treatment, and heat shock. UV irradiation selectively induces tumor cell apoptosis by causing DNA damage [47]. HOCl—a potent oxidant produced by activated neutrophils during acute inflammation—induces cell death while simultaneously enhancing immune responses, including antigen recognition, increased immune cell proliferation, and upregulated IL-2 expression [48,49]. Heat shock protocols involve sustained exposure to supraphysiological temperatures, with apoptosis induced at 41−43 °C.

2.3. Other types of lysates

Research on other types of lysates remains in its early stages, and methodological standardization for lysate preparation is still under development. However, the freeze-thaw technique has demonstrated broad applicability across diverse cell and tissue lysates, serving as a versatile and efficient platform for lysate extraction.

2.3.1. Adipose tissue lysates

Adipose tissue is a readily accessible and abundant biological resource, rich in anti-inflammatory macrophages, adipose-derived stem cells (ASCs), and a diverse array of bioactive factors (including growth factors and chemokines similar to those in platelet lysates) [50,51]. Notably, hydrogels derived from the adipose tissue extracellular matrix (ECM) exhibit sustained anti-inflammatory effects and pro-regenerative capabilities, attributed to their unique protein composition—key components include collagen, FGF-2, and TGF-β1 [52].

Collagen, a major ECM protein, self-assembles into cross-striated fibrils that serve as cell adhesion sites, enhance tissue elasticity, and improve mechanical stability. FGF-2, meanwhile, promotes cell proliferation and has demonstrated efficacy in repairing complex tissues such as the tendon-to-bone interface [53,54].

2.3.2. Osteocyte lysates

Research on osteocyte lysates originated with investigations into osteocyte-conditioned media. Early studies demonstrated that media conditioned by pulsating fluid flow (PFF) and constant fluid flow (CFF) could suppress osteoclastogenesis while increasing alkaline phosphatase (ALP) activity. These pretreatment methods result in suboptimal inhibition of osteoclast formation and insufficient bone matrix deposition, highlighting the need for more efficient strategies to augment bone regeneration [[55], [56], [57]].

Recent advances have linked interstitial fluid flow within bone tissue to osteogenic regulation. Physiological fluid shear stress activates mechanosensitive signaling pathways in osteocytes, modulating the secretion of osteogenic factors that coordinate osteoblast‒osteoclast crosstalk [58]. To replicate this phenomenon in vitro, osteocytes cultured under controlled interstitial fluid flow conditions can adopt a secretory phenotype that mimics their native microenvironment. Given that osteocyte lysates encapsulate diverse secretory proteins, their direct application presents a promising approach to bypass complex conditioning procedures.

2.3.3. Macrophage lysates

Macrophage lysates research has gained traction in oncology, particularly in the context of targeting tumor-associated macrophages (TAMs) within the immunosuppressive tumor microenvironment (TME) [59]. TAMs predominantly polarize into protumorigenic M2 phenotypes, which are associated with metastasis, whereas M1 macrophages inhibit metastasis [[60], [61], [62]]. Thus, reprogramming M2-to-M1 polarization represents a key therapeutic strategy.

Early attempts to administer M1 macrophages in vivo faced challenges: direct injection triggered lethal acute inflammation [63], whereas hydrogel-encapsulated M1 cells provoked cytokine storms due to sustained activation [64]. Lysates can also reprogram M2 macrophages toward the M1 phenotype via NF-κB define pathway activation [65], fostering antitumor immunity through the secretion of proinflammatory cytokines (e.g., IL-12, IFN-γ, tumor necrosis factor α (TNF-α), and IL-1β) [66]. Furthermore, M1 lysates enhance dendritic cell (DC) maturation and reactivate tumor-infiltrating T cells [67,68]. DCs are the most representative antigen-presenting cells (APCs) and are capable of migrating to draining lymph nodes and releasing immune regulatory signals through antigen presentation to initiate T-cell immune responses. Additionally, DCs can produce chemokines in the TME that attract T cells.

IL-12 is a proinflammatory cytokine produced by various immune cells in response to stimulation. IL-12 induces T cells and natural killer (NK) cells to produce IFN-γ, initiating additional APCs to produce IL-12, forming an effective positive feedback loop [69]. IFN-γ is a multifunctional cytokine with antiviral and antitumor properties. It plays a critical role in coordinating innate and adaptive immune responses. In inflammatory responses, it also prevents tissue damage caused by excessive immune activation [[70], [71], [72]]. IL-1β is produced mainly by stimulated immune cells and is produced at lower levels in other cell types. The binding of IL-1β to its receptor results in the recruitment of many intracellular adapter molecules, including myeloid differentiation factor 88 (MyD88) and IL-1R-associated kinase (IRAK) [73,74].

2.3.4. Bacterial lysates

Bacterial lysates contain immunostimulatory antigens used to combat infections and modulate immunity. Clinically, two types dominate: polyvalent mechanical bacterial lysates (PMBLs), which are produced via mechanical disruption, and polyvalent chemical bacterial lysates (PCBLs), which are generated through chemical lysis [75]. Compared with PCBLs, PMBLs exhibit superior efficacy in treating recurrent respiratory tract infections (RTIs), reducing acute episodes and antibiotic reliance—likely due to preserved immunogenicity [[76], [77], [78], [79]].

Beyond infectious diseases, bacterial lysates have historical significance in oncology. In 1891, William Coley pioneered the use of heat-killed Streptococcus pyogenes and Serratia marcescens mixtures (“Coley's toxins”) to treat sarcomas, carcinomas, and melanomas [80,81]. BCG is used for nonmuscle-invasive bladder cancer, leveraging bacterial lysates to stimulate antitumor immunity [[82], [83], [84]].

2.4. Reproducibility and body sensitivity of the lysates

For any therapeutic agent, the investigation of reproducibility and body sensitivity is essential. From a reproducibility perspective, platelet lysates have already entered clinical translation, with substantial results demonstrating high reproducibility. To engineer and maintain this high reproducibility, the core challenge lies in preserving the high activity of platelets before lysis and the activity of biological factors after lysis. Similarly, other types of lysates must achieve these two goals—analogous to the need to ensure the reliability of drug sources and stability of purified drugs during preparation. In existing studies, patient-derived platelet lysates are typically stored at −20 °C or −80 °C for long-term preservation; tumor cell lysates also exhibit high reproducibility, particularly autologous tumor lysates containing neoantigens—an important factor in ensuring therapeutic efficacy. Moreover, if only the activity of neoantigens is retained, these lysates may avoid side effects on normal cells and exert targeted effects on tumor cells. For other lysate types: adipose tissue lysates contain lipids in addition to proteins, and lipids are more stable than proteins; the main functional components of osteocyte and macrophage lysates are proteins; bacterial lysates may also rely on teichoic acid and lipopolysaccharides in the cell wall for activity, and low-temperature storage can maintain their stability.

Lysates can be regarded as multifactor therapeutic agents; thus, body sensitivity to lysates reflects the body's response to various biological factors, and lysate administration enables direct and rapid supplementation of these factors. These biological factors can quickly and effectively participate in regulatory mechanisms, serving as high-quality, high-quantity supplements. Notably, tumor lysates carrying neoantigens should exhibit increased sensitivity, enhancing the body's responsiveness and further improving antitumor efficacy. Adipose tissue lysates can also directly supplement collagen during wound healing. Therefore, lysates from different sources have unique mechanisms to enhance body sensitivity.

2.5. Biological sources of lysates

For platelet lysates, a systematic supply chain has been established. When used directly in clinical practice, platelet lysates are collected from blood donors and standardized by professional institutions. In relevant studies, the subjects and lysate donors are highly related (often from the same species), which may be attributed to genetic characteristics. Even for homologous genes from different biological sources, expression outcomes may vary across species, potentially leading to differences in therapeutic effects. For example, the ability of human platelet lysates to promote wound healing in mice may be inferior to that of mouse platelet lysates. This interspecies difference is also observed in adipose tissue, osteocyte, and macrophage lysates. Tumor cell lysates have more stringent requirements, as autologous resected tumor cells are often used to ensure therapeutic efficacy.

3. Preparation of the lysate-based hydrogel

A variety of materials are available for the preparation of lysate-based hydrogels, and the selection can be tailored to specific applications. With the continuous advancement of hydrogel technology, hydrogel preparation is no longer limited to single materials or methods; instead, multiple approaches complement each other and undergo continuous optimization—this represents a key focus in hydrogel design. Currently, a major limitation of traditional hydrogels is their poor mechanical properties; thus, covalent cross-linking is primarily used to enhance their mechanical stability.

3.1. Lysate-based hydrogels represented by alginate

Alginate is a hydrophilic anionic polysaccharide originally extracted from natural seaweed and bacteria [85,86]. Alginate materials are widely used in medical fields, including tissue engineering and drug delivery [87,88]. Their gel-forming ability and ion-exchange capacity make them ideal for promoting wound healing; their degradability and recyclability also render them environmentally friendly materials. The pH-dependent structure of anionic alginate, its interactions with cations, and the responsiveness of polyelectrolytes and proteoglycans enable it to deliver drugs, cells, and growth factors via electrostatic interactions. However, single-component alginate exhibits poor mechanical properties, making it difficult to withstand the physiological load of wounds and prone to deformation—thus limiting its application range for wound treatment. Therefore, other materials may need to be incorporated to improve its mechanical properties when necessary. Additionally, alginate lacks specific degrading enzymes in the body, leading to slow degradation of natural polymer chains that are not easily metabolized and excreted—potentially resulting in long-term retention in the body. The degradation of traditional ionically cross-linked alginate is affected by local ion concentrations. Although chemical cross-linking (e.g., Schiff base reaction) can improve stability, it may introduce toxic cross-linking agents (e.g., glutaraldehyde).

Alginate is an unbranched polysaccharide copolymer composed of L-guluronic acid and D-mannuronic acid linked via 1,4-glycosidic bonds [89]. Only the L-guluronic acid structure participates in covalent cross-linking with divalent cations to promote hydrogel formation [90]. The main active group of alginate is the carboxyl group, which serves as the primary site for chemical modification and cross-linking. The primary approach to preparing alginate-based lysate hydrogels is covalent cross-linking mediated by small molecules—this is also the most common and flexible method. This method does not directly link alginate and biological factors; instead, it uses a bifunctional or multifunctional cross-linking agent as a bridge to connect the two. Taking 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as an example, the reaction involves the carboxyl group of alginate and the primary amino group of biological factors. EDC activates the carboxyl group of alginate to form an unstable intermediate; subsequent addition of NHS converts this unstable intermediate into a stable amine-reactive NHS ester, which then reacts with primary amino groups on biological factors to form stable amide bonds, achieving covalent cross-linking. This reaction is relatively mature but may generate isopeptide bonds as byproducts. Cross-linking efficiency is also affected by multiple factors, which may cause aggregation and shielding of active sites.

3.2. Lysate-based hydrogels containing chitosan

Chitosan is another polysaccharide used in hydrogel preparation, with specific properties varying according to the preparation method—enabling its use for diverse purposes. In industrial production, chitosan is typically extracted from chitin via a chemical deacetylation process. The functional groups in its structure endow it with polycationic characteristics, making it suitable for the preparation of lysate-based hydrogels. In the medical field, chitosan exhibits multiple functions, including promoting wound healing, acting as a drug carrier, and regulating immunity and blood lipids. Similarly, chitosan hydrogels have certain limitations: they primarily rely on hydrogen bonding or ionic cross-linking for gel formation, and the weak natural intermolecular forces result in insufficient mechanical strength. The noncovalent network formed by physical cross-linking (e.g., with sodium β-glycerophosphate) is fragile, and chemical cross-linking may introduce toxicity. Many studies have addressed this by introducing a dual-network structure (e.g., composite cellulose nanocrystals) to strengthen the hydrogel, or by preparing hydrogels with different cross-linking ratios to determine the optimal formulation.

Chitosan is a linear polysaccharide composed of randomly distributed D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) linked via 1,4-glycosidic bonds [91,92]. The main active group of chitosan is the amino group, located on the D-glucosamine (deacetylated unit), which confers a positive charge under acidic conditions. The preparation of chitosan-based lysate hydrogels is primarily achieved via covalent cross-linking, with the Schiff base reaction being the most commonly used method. This reaction involves the amino groups of chitosan and either the amino groups of biological factors or the aldehyde groups generated after oxidation. Thus, two preparation strategies exist: if one group is an amino group, the other group needs to be oxidized to generate an aldehyde group to complete the Schiff base reaction. A reducing agent is also required to convert unstable imine bonds into stable secondary amine bonds, improving hydrogel stability. This reaction is relatively mild and can be performed at room temperature under neutral or slightly acidic conditions. However, oxidation may damage the structure of chitosan and biological factors, affecting mechanical strength and biological activity; unreduced Schiff base bonds are reversible under physiological conditions, leading to insufficient stability; and the reaction lacks specificity, potentially causing self-cross-linking.

3.3. Lysate-based hydrogels represented by methacrylated gelatin (GelMA)

GelMA is a gelatin derivative containing primarily methacrylamide groups and a small number of methacrylate groups. Gelatin is a collagen hydrolysate and the main component of the extracellular matrix in most tissues; it contains abundant arginine-glycine-aspartic acid (RGD) sequences that promote cell and factor attachment [93]. Thus, GelMA is widely used in wound repair and tissue engineering. GelMA hydrogels have several limitations: low mechanical strength, brittleness, and a requirement for light to complete cross-linking. UV light can damage biological factors and cause unnecessary loss; thus, GelMA is generally not the first choice for lysate hydrogel preparation. Visible light cross-linking systems (e.g., the blue light initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)) can be used as alternatives, but visible light curing is slow and limited in depth.

For GelMA-based lysate hydrogels, physical entrapment—a noncovalent method—is predominantly used. After mixing the lysate with GelMA, a hydrogel network is formed via photocuring, with interactions including hydrogen bonding, hydrophobic interactions, and electrostatic attraction. This method offers advantages such as simplicity and minimal chemical damage to raw materials. However, physical cross-linking is less stable than chemical cross-linking over the long term.

3.4. Lysate-based hydrogels represented by hyaluronic acid (HA)

HA is a natural unbranched polymer belonging to glycosaminoglycan heteropolysaccharides (GAGs) and is a major component of the extracellular matrix. It plays important roles in regulating angiogenesis and immunity. In solution, HA polymer chains aggregate to form a loose and elastic matrix that can support biological activities. HA hydrogels have limitations, including low mechanical strength, poor elasticity, and susceptibility to degradation by hyaluronidase in the body—potentially requiring frequent administration and increasing infection risk. Additionally, HA hydrogels exhibit weak adhesion in dynamic physiological environments, leading to rapid metabolism and unfavorable drug delivery.

HA is composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine, linked via alternating β-1,3 and β-1,4 glycosidic bonds [[94], [95], [96]]. Cross-linking strategies for HA-based lysate hydrogels include EDC/NHS reaction, Schiff base reaction, and click chemistry—the latter being an efficient and specific method. For click chemistry, HA requires premodification to introduce click groups, with main strategies including carboxyl activation and hydroxyl modification. In carboxyl activation, carboxyl groups can be converted to alkynyl or azide groups; in hydroxyl modification, epoxy reagents are used to introduce thiol or maleimide groups. This method offers advantages such as rapid reaction and low steric hindrance.

3.5. Characterization of the lysate hydrogels

Like traditional hydrogels, lysate-based hydrogels require comprehensive characterization to verify their reliability—this is an indispensable step in the preparation of any biomaterial. While the characterization of lysate hydrogels can initially follow standard protocols for traditional hydrogels, it must also incorporate analyses specific to their lysate components.

Currently, a primary goal in the lysate hydrogel field is to clarify the composition and content of various factors—this is critical for guiding disease treatment. For the large library of biological factors in lysates, these data remain scarce, with intensive detection focused on growth factors. This may be because platelet lysates are mainly used in regenerative medicine, leading to a lack of data on other factors. Thus, comprehensive detection of various factors is needed in future studies. For other cell and tissue types (e.g., tumor cells, adipose tissue, and osteocytes), current studies have focused on verifying the validity and reliability of lysates (confirming their therapeutic effects) while neglecting the detection of specific factors. This represents a gap in the field that requires further research. The lysate hydrogel field is still in its initial stage and has broad long-term development prospects. To detect the content of protein factors, the most accurate method is enzyme-linked immunosorbent assay (ELISA) to obtain specific quantitative values. To analyze the factor composition in lysates, proteomic detection (e.g., mass spectrometry (MS)-based proteomics) can be used, which can address questions related to proteins, including protein sequence and abundance levels [97]. Additionally, the release rate of lysate hydrogels after preparation can be detected, with key factors selected based on specific research objectives for targeted release rate analysis. Currently, several studies have employed these detection methods.

Electron microscopy is the most intuitive tool for analyzing lysate hydrogels. Simultaneous imaging of lysates, hydrogels without lysates, and lysate-based hydrogels allows for direct comparison. For in-depth structural analysis of lysates and lysate-based hydrogels, techniques such as infrared spectroscopy, X-ray diffraction (XRD), or nuclear magnetic resonance (NMR) spectroscopy can be used. Comparing specific functional groups and group changes before and after cross-linking provides critical references for the preparation of more optimized lysate-based hydrogels.

According to existing research, lysate-based hydrogels are not only used for drug delivery and soft tissue regeneration but also for bone repair—this imposes specific requirements on their mechanical properties. Hydrogel porosity is one key criterion: increased porosity significantly enhances water absorption and air permeability, making it suitable for scenarios requiring frequent interaction with body fluids (e.g., drug delivery, and tissue regeneration). However, high porosity also reduces hydrogel structural strength and stability, leading to deformation. For hydrogels used as supporting structures (e.g., bone repair), higher mechanical strength is required. Additionally, multiple standards can be used to characterize mechanical properties, such as stress‒strain curves, Young's modulus, and storage modulus/loss modulus (G'/G″). For wound dressing applications, hydrogel adhesion can also be evaluated; for cross-linked polymers such as lysate hydrogels, swelling ratio can be examined.

Many lysate hydrogels encapsulate cells for adjuvant therapy, with most of these cells being stem cells. Maintaining cell activity or promoting cell proliferation enhances therapeutic effects; thus, detection of cell activity or metabolic activity is necessary.

4. Application of lysate-based hydrogel

4.1. Platelet lysate-based hydrogels

Two principal strategies exist for formulating platelet lysate-based hydrogels: (1) postsynthesis incorporation of lysates into a preformed hydrogel matrix; (2) direct dissolution of hydrogel precursors in platelet lysates to create a fully integrated system (Fig. 4).

Fig. 4.

Two approaches for preparing lysate-based hydrogels. (A) Lysates were mixed with the base hydrogel to obtain the complete hydrogel. (B) Directly adding the crosslinking agent to lysates to obtain the complete hydrogel.

For the postsynthesis incorporation strategy, a study developed a nanocrystalline cellulose-chitosan hydrogel via acetic acid/sodium hydroxide treatment, achieving comparable therapeutic outcomes upon lysate integration. In contrast, conventional hydrogels utilizing tannic acid and quaternized chitosan (QCS) require laborious extraction and chemical modification steps (e.g., QCS synthesis for neutral solubility) but exhibit inferior performance relative to platelet lysate-based variants [98,99].

The direct dissolution strategy maximizes lysate concentration and therapeutic potential. Zhang et al. [100] incorporated human platelet lysate (PL) directly into the GelMA prepolymer solution and fabricated PL‑functionalized GelMA hydrogel microspheres via photocrosslinking. The sustained release of multiple growth factors abundant in PL significantly promoted the proliferation, migration, and odontogenic differentiation of encapsulated hDPSCs, while also enhancing angiogenesis and pulp‑like tissue regeneration. Tibourtine et al. [101] synthesized an aerogel by formulating a base hydrogel in platelet lysate, followed by solvent exchange (with acetone) and supercritical CO₂ drying. The resulting scaffold supported robust growth of BALB-3T3 fibroblasts and human umbilical vein endothelial cells (HUVECs), underscoring its utility in tissue engineering. Marfoglia et al. [102] developed a photocrosslinked GelMA/alginate hydrogel by dissolving polymers directly in lysates with LAP-induced gelation. Notably, platelet lysates outperform fetal bovine serum (FBS) in supporting cellular proliferation and differentiation; recent studies have highlighted the superior osteogenic potential of bone marrow mesenchymal stem cells in lysate-based media. Another study advanced this paradigm by creating a tyramine-crosslinked hyaluronic acid hydrogel reconstituted in lysates with horseradish peroxidase/H₂O₂ to enhance chondrogenesis [103,104].

These advancements position platelet lysate-based hydrogels as promising alternatives to traditional biomaterials, offering enhanced bioactivity and streamlined fabrication.

4.2. Tumor lysate-based hydrogels

The fabrication of tumor lysate-based hydrogels parallels that of platelet lysate-based hydrogels, employing either postsynthetic incorporation or direct dissolution of hydrogel precursors in lysates. For solvent-free applications, self-assembling peptides are frequently utilized owing to their tunable mechanical properties, compatibility with bioactive motifs, and capacity to integrate extensive antigen libraries—features that have enabled their broad application in tissue engineering, drug delivery, and biosensor development [105,106].

Current research emphasizes melanoma—a readily accessible cutaneous malignancy—as a model system. Yang et al. [107] engineered a melittin-RADA32 peptide hydrogel incorporating cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN, a TLR9 agonist) and tumor lysates. CpG-ODNs activate TLR9-expressing immune cells (e.g., plasmacytoid dendritic cells), resulting in potential melanoma immunotherapy outcomes. Similarly, Song et al. [108] developed a methoxy polyethylene glycol-block-poly(L-valine) (mPEG-b-PLV) copolymer hydrogel loaded with lysates and poly(I:C), which activated dendritic cells and amplified cytotoxic T lymphocyte responses against melanoma.

For the direct lysate solvent strategy, Chen et al. [109] formulated a hydrogel using oxidized sodium alginate (OSA)-modified tumor lysates as a solvent, augmented with nicotinamide riboside (NR) and the glycogen synthase kinase 3 (GSK-3) inhibitor SB415286. Calcium chloride facilitated hydrogel formation, significantly enhancing T-cell-mediated immunity in melanoma models.

In contrast, conventional solvent-free hydrogels for melanoma treatment often incorporate chemotherapeutic agents such as doxorubicin (DOX); despite their efficacy, these agents increase the risk of myelosuppression and cardiotoxicity [110].

4.3. Other lysate-based hydrogels

4.3.1. Adipose tissue lysate hydrogels

Wang et al. [9] engineered an adipose tissue lysate-based hydrogel via low-temperature gelation, directly dissolving alginate linkers in lysates with EDC and NHS as cross-linking agents. At −20 °C, carboxyl groups on alginate covalently bonded with primary amine-containing proteins, facilitating hydrogel formation. This system exhibited prolonged immunomodulatory activity and enhanced spinal cord injury regeneration.

4.3.2. Osteocyte lysate hydrogels

Zheng et al. [111] developed an osteocyte lysate-based hydrogel activated by oscillating fluid flow; this system stimulated osteocyte secretion of soluble mediators to promote osteoblast proliferation/differentiation while suppressing osteoclast activity. Enhanced glycolysis and activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) and Wnt/β-catenin pathways further improved osteogenic efficacy. This approach mimics physiological bone-forming microenvironments, enabling rapid osteocyte adaptation—a notable advantage over conventional photothermal hydrogels.

4.3.3. Macrophage lysate hydrogels

M1 macrophage lysates (typically polarized with IFN-γ and lipopolysaccharide (LPS)) are cross-linked via EDC/NHS chemistry into sodium alginate matrices for tumor therapy applications [112]. Li et al. [113] advanced this strategy by cocrosslinking tumor lysates and M1 macrophage lysates at −20 °C, achieving synergistic antitumor effects through cold gelation. Unlike chemotherapeutic-loaded systems (e.g., Luo et al. [114] used an oxaliplatin-conjugated G5 polyamidoamine/dextran hydrogel, which is prone to neurotoxicity), lysate-based hydrogels avoid long-term toxicity concerns—positioning them as safer therapeutic alternatives.

4.3.4. Bacterial lysate hydrogels

Building on the century-old use of attenuated Mycobacterium bovis (BCG) in tuberculosis vaccines, Mirela et al. synthesized a poly (lactic-co-glycolic acid) (PLGA) triblock copolymer hydrogel within BCG lysates; this platform enhanced cathepsin S (CTSS)-mediated antigen processing. Pathogens and the human microbiome represent underexplored reservoirs of commensal and pathogenic species—their lysates could unlock novel therapeutic paradigms in immunotherapy and regenerative medicine.

5. Conclusion and future prospects

When lysates are used as therapeutic components—either directly incorporated into hydrogels or as solvents to dissolve hydrogel materials—they offer several advantages over traditional hydrogels, including simpler preparation, lower costs, ease of clinical translation, broad availability of raw materials, versatile therapeutic effects, minimal loss of active components, and high reproducibility. Based on lysate sources, lysate-based hydrogels can be categorized as follows: (1) Platelet lysate-based hydrogels: Platelet lysates contain a wide range of bioactive factors (e.g., cytokines, chemokines, transport proteins, protease inhibitors, growth factors, plasma-derived proteins—including G-CSF, CCL5/RANTES, transferrin, α1-antitrypsin, IGF-1, and albumin), endowing them with versatile therapeutic benefits and promising prospects. (2) Tumor lysate-based hydrogels: Tumor cells—especially those derived from autologous resected tumors—carry unique antigen fragments resulting from mutations. Leveraging the immune system, these lysates enable more targeted and effective tumor treatment. Beyond hydrogel preparation, tumor lysates also advance tumor vaccine research, as their preparation and research and development align with vaccine production processes. (3) Other lysate-based hydrogels: These have been less studied in the context of hydrogels, indicating significant potential for future research. Adipose tissue, rich in collagen and FGF-2, is widely used in tissue repair and cosmetic industries; osteocyte lysates can induce osteoblast proliferation/differentiation while inhibiting osteoclasts to promote bone regeneration; macrophage lysates can drive M1 polarization (enhancing immunogenicity); and bacterial lysates show potential for treating various bacterial infections.

After discussing lysate sources, this review summarized preparation methods. The freeze‒thaw cycle is widely accepted for lysate preparation, offering a simple and universal approach; after freeze-thawing, lysates can be directly used for hydrogel preparation. Additionally, different lysate sources have unique preparation methods: platelet lysates can also be prepared via solvent/detergent inactivation, ultrasonication, or calcium chloride treatment; tumor lysates can be prepared via UV irradiation, hypochlorous acid treatment, or heat shock. This review also discussed lysate reproducibility, sensitivity, and biological sources. For reproducible bioactive substances, the key to ensuring reproducibility is maintaining cell/tissue activity before lysis and biological factor activity after lysis—goals that are readily achievable, facilitating clinical translation of lysate-based hydrogels. The diverse biological factors in lysates ensure their sensitivity, and each lysate type has characteristic components that enhance the body's responsiveness.

This review also detailed lysate-based hydrogel preparation methods, highlighting key principles, advantages, disadvantages, and relevant characterization techniques. These characterization methods span multiple levels, enabling multidimensional analysis of lysate-based hydrogels. Critically, detecting the specific content of each factor (or at least several factors relevant to the study) is essential; electron microscopy and spectral data reflect the intrinsic properties of lysate-based hydrogels; and if cell loading is required for disease treatment, cell activity detection after coculture is necessary. Finally, application examples were provided: the core concept of lysate-based hydrogel preparation is either post-incorporation of lysates or direct use of lysates as solvents to dissolve hydrogel materials. Using lysates as solvents maximizes therapeutic component concentration, optimizing efficacy—for example, mixing tumor cell lysates with M1 macrophage lysates. The diversity of lysate sources enables versatile therapeutic effects of hydrogels.

The choice of cross-linking agent and conditions also affects therapeutic efficacy; however, lysis degree and cross-linking degree are critical factors requiring further discussion. The key is to maximize the preservation of active components, which depends on two aspects: (1) Lysis degree: Lysates must be sufficiently disrupted to fully release active components, but over-lysis should be avoided to prevent component degradation (e.g., temperature conditions during freeze‒thawing inevitably cause protein degradation). (2) Cross-linking degree: The selection of cross-linking agents and conditions requires further exploration. These two aspects may be focused areas for future research. Directly using lysates as solvents to dissolve hydrogel materials may be a superior approach. Studies have also supplemented lysates with various components to optimize effects or address more challenging diseases: for example, combining platelet lysates with the MMP-13 inhibitor 5-bromindole-2-carboxylic acid (BICA) to inhibit chondrocyte hypertrophy and promote chondrogenesis [115,116]; combining tumor lysates with the TLR3 agonist poly(I:C) to enhance immune responses; and combining osteocyte lysates with oscillating fluid to simulate the osteogenic environment, inhibit osteoclasts, and activate osteoblasts—enhancing osteogenic effects.

Currently, the lysate-based hydrogel field faces challenges. First, existing studies lack data on the specific factor contents in lysate-based hydrogels, leading to a degree of research blindness. Providing specific data would enable more meaningful comparisons with traditional hydrogels and offer strong references for other researchers—undoubtedly advancing the entire field. Second, coordinating the numerous biological factors in lysates is a key challenge. For example, platelet lysate-based hydrogels deliver a large “library” of biological factors to wounds, providing molecules required for all wound healing stages; however, not all factors are beneficial, and potential antagonism (e.g., IL-1 plays a critical role in the inflammatory phase but may antagonize other factors in later stages, impairing therapeutic effects) may occur. Thus, effectively coordinating factor interactions requires resolution. The complexity of lysate factors currently hinders field development; since biological factors are inherently present in cells/tissues and difficult to remove, a practical solution is to modify hydrogel preparation strategies—using hydrogel materials to modulate the activity of different biological factors in lysates. This is also an advantage of lysate-based hydrogel preparation: high operability enables coordination of biological factor interactions.

Owing to the broad-spectrum therapeutic potential of hydrogels, numerous future research directions exist. First, platelet lysate-based hydrogels—rich in growth factors—exhibit strong tissue regeneration capabilities, with potential applications in wound healing, diabetic ulcers, burns, spinal cord injury (SCI) treatment, peripheral nerve repair, injectable formulations for myocardial infarction repair, and coculture with endothelial progenitor cells (EPCs) to promote microvascular network formation (enabling artificial blood vessel development). Second, chimeric antigen receptor T-cell (CAR-T) and CAR-natural killer (CAR-NK) therapies are emerging tumor treatments; tumor lysate-based hydrogels can simulate the TME to preactivate cells and leverage neoantigens to attract cells and enhance solid tumor recognition. Combining lysate-based hydrogels with CAR-T/CAR-NK therapies for in vivo delivery can reduce cytokine storm risk. Third, adipose tissue lysate-based hydrogels contain TGF-β3, which reduces fibrosis—offering potential for burn treatment and skin elasticity enhancement [117]. Adipose tissue lysates also show promise in medical aesthetics: their abundant collagen can stimulate autologous collagen production (a research hotspot in medical aesthetics) for use as regenerative dermal fillers, and combining with phototherapy can improve skin firmness; fat-related lysates may also address obesity, though specific mechanisms require clarification. Of course, other lysate-based hydrogels likely have undiscovered functional potential; however, current research covers only a small subset of lysate types, necessitating further exploration to expand their applications. From the broader perspective of biomedical engineering, lysate-based hydrogels represent a disruptive innovation. Current studies focus primarily on therapeutic effects; with deeper future research, lysate-based hydrogels may permeate all levels of hydrogel applications in bioengineering.

In summary, further exploration of diverse lysate sources or functionalization of existing sources, development of optimal lysis and cross-linking conditions, active combination of lysates with other effective components, and coordination of biological factor interactions will continue to drive this field toward broader and deeper development.

CRediT authorship contribution statement

Yang Lv: Writing – review & editing, Writing – original draft. Siteng Tieng: Conceptualization. Xiaoling Xu: Conceptualization. Wei Chen: Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.