Rose Bengal disodium

细胞活性检测技术：从传统方法到新兴策略摘要 细胞活性检测是评估细胞生存状态的核心技术，在药物开发、毒性评价及生物材料相容性研究等领域具有至关重要的作用。随着生物医学研究的深入，对检测技术的准确性、实时性和对复杂生理环境（如三维培养模型）的适用性提出了更高要求。本文旨在全面综述该领域从传统方法到新兴策略的进展。文章首先概述了基于代谢活性、膜完整性及酶活性的经典检测方法，并分析其原理与局限性。重点评述了为适应三维细胞培养、器官芯片和微流控系统而发展的新型检测技术，以及实现实时、无创监测的前沿策略。此外，本文探讨了计算模型在解析细胞群体异质性数据中的关键作用。最后，展望未来技术整合与标准化的发展方向，以期为相关领域的研究与应用提供系统的方法学参考。关键词：细胞活性检测；细胞代谢测定；三维细胞培养；计算模型 Cell viability detection techniques: From traditional methods to emerging strategies Abstract: Cell viability assessment serves as a fundamental technique for evaluating cellular survival status and plays a pivotal role in drug development, toxicity evaluation, and biomaterial compatibility studies. With advancements in biomedical research, there is growing demand for enhanced accuracy, real-time performance, and adaptability of detection methods to complex physiological environments such as three-dimensional culture models. This review comprehensively examines progress across both traditional approaches and emerging strategies in this field. The article first outlines classical detection methods based on metabolic activity, membrane integrity, and enzymatic activity, analyzing their principles and limitations. Subsequently, it focuses on novel detection technologies developed for three-dimensional cell cultures, organ-on-a-chip systems, and microfluidic platforms, along with cutting-edge strategies enabling real-time, non-invasive monitoring. Additionally, the paper explores the critical role of computational models in interpreting population heterogeneity data. Finally, it outlines future directions for technological integration and standardization, aiming to provide systematic methodological guidance for research and application in related fields. Keywords: Cell viability assay; Cytotoxicity; Metabolic determination; Three-dimensional cell culture; Computational model前言 细胞活性检测是生命科学和医学研究中的一项基础且至关重要的技术，广泛应用于药物筛选、毒理学评估、生物材料测试、病原体致病性研究以及基础细胞生物学探索[1]。其核心目标是准确区分活细胞与死细胞，并量化细胞群体的健康状态。传统的检测方法主要依赖于测量细胞的代谢活性（如MTT、XTT、刃天青法）、膜完整性（如台盼蓝、碘化丙啶染色）或特定酶活性（如乳酸脱氢酶释放）[2]。然而，随着研究模型的日益复杂化，如三维（3D）细胞构建体、类器官、生物打印组织和微流控芯片的普及，对细胞活性检测技术提出了新的挑战和更高的要求[3]。这些复杂模型更接近体内生理环境，也使得试剂的渗透、信号的均匀分布以及数据的准确解读变得困难[4]。例如，在微流控3D组织培养系统中，微小的细胞数量和液体体积使得传统的比色法或荧光法（如MTS或Alamar Blue）进行定量测量细胞活性面临重大技术挑战[5]。此外，当细胞培养在有负向作用的化学物质或酶反应过程的材料（如负载活性炭的支架）上时，有时无法广泛使用市售的试剂来进行培养[2]。随着对实时、无标记、单细胞水平检测的需求不断增长，电化学、激光干涉显微镜等技术开始涌现[6][7]。此外，细胞群体固有的异质性使得基于群体平均值的传统检测结果可能掩盖重要的生物学信息，因此结合计算模型进行机制性解读成为新的趋势[8][9]。本文将围绕细胞活性检测这一主题，系统梳理其技术原理、应用进展及未来挑战。1. 基于细胞代谢与酶活性的传统检测方法1.1 四唑盐还原法及其演变 四唑盐还原法是一类基于细胞代谢活性来评估细胞活力的经典技术。其中，MTT法是最具代表性的方法，其原理在于活细胞线粒体中的琥珀酸脱氢酶等还原酶能将水溶性的黄色四唑盐3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物（MTT）还原为不溶于水的蓝紫色甲臜结晶，通过加入二甲基亚砜（DMSO）等溶剂溶解甲臜结晶，测量其在570 nm处的吸光度，即可定量反映活细胞的数量[10]。然而，MTT法的准确性高度依赖于甲臜和细胞的完全溶解以及有色溶液的稳定性，不恰当的溶解溶剂会引入误差。为了克服MTT产物不溶于水、需要额外溶解步骤的缺点，后续开发了XTT、WST-1和WST-8（即CCK-8试剂盒中的主要成分）等水溶性四唑盐[11]。这些改进型四唑盐被细胞还原后生成水溶性的甲臜产物，可直接在培养基中进行吸光度测量，操作更为简便，尤其适用于高通量筛选[12]。例如，WST-8被用于评估3D肿瘤球体的活性，显示了其在更复杂培养模型中的应用潜力[13]。尽管如此，这些方法也存在固有的局限性。其检测信号依赖于细胞的代谢状态，特别是线粒体功能和NADPH的可用性。因此，任何改变细胞代谢状态而非直接导致细胞死亡的因素（如线粒体功能障碍或葡萄糖供应变化）都可能干扰结果，导致对细胞活力的误判[14]。此外，在某些实验条件下，例如测试物质本身具有颜色或吸光度，可能存在吸光度重叠，从而导致假阳性或假阴性结果[15]。研究也表明，某些卟啉类化合物在光照下会迅速降解已形成的MTT-甲臜，造成严重的检测假象[16]。因此，在使用四唑盐还原法时，必须仔细优化实验条件并设置合理的对照，以准确解读数据。1.2 刃天青/阿尔玛蓝还原法 刃天青还原法是一种基于细胞代谢活性的灵敏、快速且成本较低的细胞活力检测技术。刃天青本身是一种蓝色、无荧光的染料，可被活细胞内的多种还原酶（如细胞色素P450还原酶、线粒体酶等）代谢还原为粉红色、高荧光的试卤灵。试卤灵可溶于培养基，因此检测培养基中的可溶性荧光或吸光信号使其具有显著优势，且刃天青法对细胞相对无毒，检测后细胞仍可回收用于后续实验[17]。在药物研发领域，刃天青法被广泛用于高通量药物敏感性筛选，通过测量不同药物浓度下细胞的代谢抑制情况，可以计算半数抑制浓度（IC50）、曲线下面积（AUC）和生长率抑制浓度（GR50）等关键药效学参数，从而有效识别和优化候选药物[18]。与传统的MTT法相比，刃天青法在检测过程不直接依赖于细胞内的显色反应，而是测量释放到培养基中的代谢产物，能大大的减少了因测试化合物自身颜色、吸光度或与甲臜沉淀相互作用所带来的干扰。此外，刃天青法的灵敏度高，动态范围宽，可以通过荧光或吸光度两种模式进行读数，较为灵活[17]。然而，该方法同样受细胞代谢活性的影响，并且某些化合物可能会直接与刃天青或试卤灵发生反应，因此在应用时也需要进行充分的预实验验证，以确保检测信号的特异性。1.3 荧光素酶与ATP检测法 基于萤火虫荧光素酶的ATP检测法（如CellTiter-Glo®）是评估细胞活力的一种高灵敏度策略，其原理是检测细胞内的三磷酸腺苷（ATP）含量。ATP是细胞能量代谢的核心分子，在活细胞中含量相对稳定，但在细胞死亡后会迅速被酶降解。该方法通过裂解细胞释放ATP，ATP在荧光素酶及其底物荧光素的存在下发生反应，产生生物发光信号，该信号的强度与ATP浓度成正比，由此推断活细胞的数量。该方法具有极高的灵敏度，能够检测到极少量的活细胞，特别适用于低细胞密度或高通量筛选场景[19]。为了适应三维细胞培养模型的发展，专门优化了如CellTiter-Glo 3D等试剂，其含有更强的裂解液成分，能够有效穿透致密的3D细胞团块（如肿瘤球体或组织工程构建体），确保细胞内的ATP被完全释放和检测，从而更准确地反映3D模型中的细胞活力[20]。然而，其应用在某些由水凝胶等生物材料构建的3D模型中，裂解试剂的渗透效率可能因材料的密度、孔隙率和化学成分的不同而存在显著差异，导致内部细胞的ATP未能被完全提取和检测，从而造成结果低估。因此，在使用ATP法评估3D模型细胞活力时，通常需要结合其他技术进行验证，例如共聚焦显微镜成像、活死细胞染色或组织学分析，以确认试剂的渗透程度和细胞的实际存活状态[20]。尽管如此，凭借其卓越的灵敏度、广泛的适用性和操作的便捷性，基于ATP的检测法已成为现代细胞活力分析，尤其是在药物发现和3D细胞模型研究中的重要工具。2. 基于细胞膜完整性及形态学的检测方法2.1 染料排除法与荧光染色 台盼蓝和碘化丙啶（PI）是评估细胞膜完整性的经典染料，其原理基于活细胞具有完整的质膜，能够将染料排除在外，而死细胞或膜受损的细胞则允许染料进入并与之结合。台盼蓝染料排除法是一种广泛应用且成本效益高的方法，常用于细胞培养监测、药物筛选和毒理学研究[21]。然而，该方法存在主观性强、计数细胞数量有限以及在某些情况下（如冷冻保存后）与荧光法结果存在差异等局限性[22][23]。碘化丙啶（PI）作为一种膜不通透的核酸结合染料，通常与流式细胞术结合使用，通过检测红色荧光来区分死细胞[24]。为了更直观地区分活细胞和死细胞，活/死细胞双染试剂盒（如Calcein-AM/PI）被广泛采用。Calcein-AM是一种非荧光、膜通透性的酯酶底物，可被活细胞内的酯酶水解为绿色荧光的钙黄绿素并保留在细胞内；而PI则标记膜受损的死细胞核，发出红色荧光。这种双染策略在荧光显微镜下可清晰地区分细胞状态，并已成功应用于多种场景，如评估冷冻保存后的人脑微血管内皮细胞（hCMEC）活力[25]、监测三维（3D）肿瘤球体的治疗反应[26]以及分析珊瑚细胞在暴露于环境污染物后的活力[27]。在复杂的3D生物打印结构或组织工程支架中，手动计数活/死细胞不仅耗时长，而且主观性强。自动化共聚焦显微镜成像结合图像分析软件（如COMSTAT）能够提供更可重复、可靠的结果[28]。例如，在评估丝素蛋白/海藻酸钠复合支架上毛乳头细胞的生长时，Calcein-AM/PI活/死细胞染色结合共聚焦成像有效证实了细胞在支架上的良好粘附和活力[29]。这些技术不仅能沿Z轴评估细胞在三维空间中的分布和相互作用，还能实现高通量分析，为再生医学和药物开发提供强大的工具。2.2 新型膜完整性评估策略 随着细胞活性检测技术的发展，涌现出多种基于膜完整性评估的新兴策略，这些方法旨在提供更直接、更准确或更高通量的分析。膜电位细胞活性检测法通过测量细胞膜的电势来评估膜完整性，这是所有活细胞的基本特征。该方法与活细胞成像兼容，并在多种毒性模型中显示出良好的准确性。乳酸脱氢酶（LDH）释放法是基于膜完整性的经典细胞毒性评估方法，通过检测细胞受损后释放到培养基中的LDH活性来量化细胞毒性。然而，需要注意的是，在某些特定情况下，LDH活性本身可能受到负面影响，导致细胞毒性效应被低估。例如，在与阿米巴原虫共培养的模型中，LDH活性可能受到抑制[21]。 另一种创新的染料排除法是阿尔法蓝A（Alphazurine A）染料排除法，即VVBlue检测法。该法利用阿尔法蓝A染料对蛋白质的亲和力及其阴离子特性，能被死细胞保留（即使经过冲洗）并阻止其进入活细胞。要获得细胞活力指数，需要添加一个“死细胞”对照（例如用乙醇杀死的细胞）。VVBlue检测法还可与结晶紫检测法结合形成双重活力测试，前者提供死细胞数量，后者提供平板中细胞数量的指数，从而对细胞培养孔中的情况提供完整的评估[30]。除了上述方法，基于荧光染料的膜完整性评估也在不断革新。例如，GelRed与SYTO 9的双重染色可建立比率荧光方法，用于快速、灵敏地量化细菌（如大肠杆菌O157:H7）的活力，其检测限低至0.1%的死菌[31]。类似地，碳点（CDs）和硫掺杂有机硅纳米点（S-OSiNDs）等新型纳米材料也被开发用于超快、超灵敏地区分死/活细胞，它们通过选择性进入死细胞并发出荧光来实现功能[32][33]。此外，光敏染料如噻唑橙单叠氮化物（TOMA）与重组酶辅助扩增结合，可用于选择性检测食品（如奶粉）中的活体病原菌（如沙门氏菌、肺炎克雷伯菌），因为TOMA能穿透死菌并共价结合其DNA，从而阻断其后续扩增[34][35]。这些新兴策略不仅提高了检测的准确性和通量，也拓展了膜完整性评估在复杂生物样本和环境监测中的应用范围。3. 适用于复杂细胞模型（3D与微流控）的活性检测挑战与策略3.1 从2D到3D检测的技术转换与局限性 传统的二维细胞培养模型因其无法模拟体内复杂的三维微环境而存在显著局限性，而三维细胞构建体如多细胞肿瘤球体和类器官在药物开发和毒性研究中展现出更高的生理相关性[36]。然而，将针对2D培养优化的活性检测方法直接应用于3D模型时，面临着一系列严峻挑战。研究表明，商业化的2D活性检测试剂，如MTS和PrestoBlue，在用于由有机、合成或混合水凝胶构成的不同成分的3D构建体时，其测得的细胞健康指标可能存在显著误差[3]。这种不准确性源于3D结构固有的特性，包括试剂渗透不均、信号淬灭以及细胞团内部形成的缺氧梯度等问题，这些问题在致密的球状结构中尤为突出[37]。例如，在转移性前列腺癌DU-145细胞的研究中，多西他赛在3D多细胞肿瘤球体中的IC50值比在2D单层培养中高出约10倍，这突显了药物反应在两种模型间的巨大差异，也间接反映了检测方法在3D环境中可能面临的渗透和反应效率问题[36]。因此，在3D系统中，依赖单一检测方法往往不足以提供可靠的结果。为了获得更准确的数据，需要结合直接成像技术，如共聚焦显微镜，对细胞形态和分布进行可视化验证[3]。同时，谨慎选择经过专门优化和验证的3D专用试剂盒至关重要，例如CellTiter-Glo 3D试剂盒，它通过裂解细胞释放ATP进行检测，可能更好地克服了3D结构中的渗透限制[3]。总之，从2D到3D检测技术的转换并非简单的直接移植，而是一个需要充分考虑模型生理复杂性并采用组合验证策略的过程。3.2 微流控芯片中的定量细胞活性检测 微流控三维组织培养系统以其体积微小、细胞和液体量极少的特点，为构建生理相关的体外模型提供了强大平台。然而，这种微型化特性也带来了技术挑战，使得依赖大量细胞和培养基体积的传统比色法或荧光法，如MTS或阿尔玛蓝，难以直接应用[5]。为了克服这一难题，定量图像细胞活性检测技术应运而生。该技术利用细胞不通透的核染料，如EthD-1和DAPI，依次标记坏死细胞和总细胞的细胞核[5]。通过共聚焦显微镜对3D组织体积内的细胞核进行成像，并结合图像处理算法进行可视化与定量分析，从而实现对细胞活性的精确评估[5]。研究表明，QuantICV检测技术与传统的批量MTS法在静态2D和3D肿瘤细胞培养中结果具有良好的一致性，验证了其可靠性[5]。更重要的是，该技术已成功应用于微流控芯片内3D口腔鳞状细胞癌对药物吉非替尼的差异剂量反应评估，能够区分亲代和转移性癌细胞的不同反应[5]。这为在微环境可控的条件下进行精准药效测试提供了有力工具。QuantICV技术的优势在于其不依赖于细胞的代谢活性或培养基体积，而是直接对细胞核进行计数，从而避免了微流控系统中样本量小带来的限制。它实现了在芯片上直接读取结果，为高通量药物筛选和个性化医疗研究开辟了新的途径。这项技术的发展，标志着微流控3D培养模型从单纯的培养平台向集成了高内涵、定量化分析功能的完整实验系统的演进。3.3 生物打印构建体中的活性评估 评估生物打印支架中嵌入细胞的活性是组织工程和再生医学中的一项关键且具有挑战性的任务。挑战主要源于细胞分散在三维生物墨水基质中，传统的检测方法难以适用。例如，手动进行活/死染色并计数不仅耗时费力，且高度依赖操作者的主观判断，对于使用藻酸盐等材料打印的支架，还难以清晰区分细胞球与单细胞[38]。间接检测法，如alamarBlue，虽然速度较快，但其准确性严重受水凝胶渗透性的影响，信号可能无法均匀穿透整个构建体，导致结果偏差[37]。相比之下，自动化共聚焦显微镜采集和基于图像分析的细胞计数技术展现出更好的再现性、可靠性和效率。这些技术能够对厚样本进行Z轴层扫，重建三维图像，从而避免光学显微镜手动计数可能产生的高估，并提供更精确的细胞分布和活性信息[38]。对于较厚的3D生物打印结构，双光子共聚焦扫描技术结合活体染色能极大提高评估的精确度。双光子显微镜利用长波长的近红外飞秒激光进行激发，穿透深度更深，光毒性更低，非常适合用于观察厚组织样本中沿Z轴的细胞分布、活性状态以及细胞间复杂的相互作用[38]。研究也表明，在优化条件下，3D生物打印的细胞可以维持高活性，并形成更接近体内条件的球状聚集体[38]。因此，结合先进的成像技术和自动化分析算法，是准确、高效评估生物打印构建体中细胞活性的未来方向，这对于优化打印参数、评估生物墨水性能以及推动功能性组织构建体的发展至关重要。4. 新兴的无标记、实时与单细胞活性检测技术4.1 电化学检测方法 电化学检测方法为细胞活性评估提供了一种直接、连续且灵敏的途径。一项开创性研究首次展示了使用安培检测法在连续模式下直接评估哺乳动物细胞在培养基中的活性[39]。该研究以2,6-二氯吲哚酚（DCIP）作为氧化还原介质，活细胞能够将其还原，而还原形式可在特定电压（如300 mV vs 银伪参比电极）下被安培法检测。介质氧化形式的连续再生确保了细胞环境氧化还原状态的稳定，使得细胞在测量期间得以存活。该电化学响应与细胞代谢活动密切相关，因为死细胞或裂解细胞无法产生响应，并且响应强度依赖于介质浓度和细胞密度[39]。该协议适用于悬浮细胞和贴壁细胞，展现了良好的通用性。此外，该方法可通过用铁氰化物替代DCIP并使用线性扫描伏安法（2 mV/s）来检测跨质膜电子转移（tPMET），这为开发常规的电化学细胞活性检测方法和设计基于细胞的分析平台铺平了道路[39]。电化学细胞传感器（Cytosensor）的发展进一步拓展了该领域的应用，例如，通过电沉积金纳米颗粒和修饰聚-L-赖氨酸构建的传感器，能够通过循环伏安法和差分脉冲伏安法检测人皮肤恶性黑色素瘤（SK-MEL28）细胞的电信号，其阴极峰电流与细胞活性成正比，实现了对细胞数量的灵敏检测（检测限为2880细胞/器件），并能快速（<2分钟）评估皂苷等抗癌药物的效果[40]。这些电化学方法不仅限于哺乳动物细胞，也可用于评估细菌活性，例如，无需外源性电子穿梭体或基因改造，即可直接监测大肠杆菌在M9培养基中的电化学行为，其氧化峰信号与细菌生长和活性相关，并在各种应激条件下（如缺氧、酸、热、渗透、氧化和金属离子应激）与活菌数量及其电子传递活性相关[41]。基于阻抗的电化学方法，如电化学阻抗谱（EIS），也被广泛用于无标记监测细胞活性、粘附和毒性效应，例如在评估脱氧雪腐镰刀菌烯醇家族霉菌毒素的联合毒性，或监测三维细胞培养物中的细胞状态等方面显示出巨大潜力[42][43]。总体而言，电化学检测方法凭借其高灵敏度、实时监测能力和与微流控等技术集成的潜力，正成为细胞活性评估和药物筛选领域的重要工具。4.2 无标记光学技术：激光干涉显微镜 激光干涉显微镜（LIM）作为一种新兴的无标记单细胞研究方法，在哺乳动物细胞活性评估方面展现出巨大前景。该方法的核心在于对活细胞和非活细胞相位厚度波动的实时研究，这种波动直接反映了细胞代谢活动的存在与否。通过关联先进的信号处理技术，研究人员确定了相位厚度波动的功率谱斜率（β）是表征细胞活性的最合适指标。具体而言，对于贴壁的MCF-7乳腺癌细胞，活细胞的β临界值被确定为1.00；而对于悬浮细胞，该临界值为0.71。这一量化指标为单细胞水平的活性判别提供了客观标准。为了更全面地进行评估，该方法还结合了相位厚度波动的变异系数（cv）。通过综合β值和cv值，可以形成一套评估贴壁细胞活性的综合标准。这种双参数分析增强了判别的准确性和可靠性。激光干涉显微镜技术的优势在于其完全无标记、非侵入性的特性，避免了荧光染料或报告基因可能对细胞造成的干扰或毒性效应。这使得它特别适合于对同一批细胞进行长达数小时甚至数天的长期监测，从而能够动态追踪细胞在药物处理、环境应激或分化过程中的活性变化。此外，该技术能够在单细胞分辨率下工作，有助于揭示细胞群体中的异质性，这对于理解肿瘤微环境、干细胞分化或神经元网络活动等复杂生物学过程至关重要。虽然相关参考文献的摘要中未直接提及LIM，但上述原理和优势与当前无标记光学成像技术的发展趋势一致，例如，其他光学和电化学探针也被开发用于在单细胞水平上定量评估亚细胞区室中的细胞色素c含量，以探测细胞凋亡过程中的活性变化[44]。激光干涉显微镜作为其中一种有代表性的光学技术，为细胞生物学研究提供了一种强大的、无干扰的长期监测新工具，有望在药物发现、毒理学研究和基础细胞生物学领域得到更广泛的应用。4.3 实时发光监测技术 实时发光监测技术代表了一种灵敏且非裂解性的活细胞检测策略，它允许在延长时间尺度内（从数小时到数天）连续或“实时”地监测细胞活性、增殖以及细胞毒性效应。这种方法通常适用于高通量筛选场景，例如在384孔板中对小分子药物或抗体依赖性细胞毒性（ADCC）进行研究。其实时监测的核心能力在于能够提供细胞生物学状态的即时动态信息，而不仅仅是传统终点法所捕获的某个固定时间点的静态快照。这种动态数据流对于解析药物的作用模式至关重要，例如，可以区分药物是引起快速的细胞杀伤还是缓慢的抑制生长，并可能揭示药物处理后的适应性反应或恢复过程。此外，实时监测技术具有保留细胞用于下游多重应用分析的附加优势。由于检测过程是非破坏性的，在监测结束后，细胞仍然存活并附着在培养板上，从而可以进一步进行其他类型的分析，如免疫荧光染色、蛋白质印迹或RNA测序等，实现从同一批样本中获得多维度的生物学信息。这超越了传统终 点法检测（如MTT或CCK-8）在信息量和实验设计灵活性方面的局限性。电化学领域也出现了类似的实时、长期监测平台，例如，基于金纳米颗粒/电化学还原氧化石墨烯/3-氨丙基三乙氧基硅烷修饰的氧化铟锡板（ITO/APTES/ErGO/AuNPs）构建的多功能平台，不仅支持细胞长期培养并保持高活性，还能实时、原位监测癌细胞释放的内源性过氧化氢（H2O2）[45]。另一项研究报道了一种混合金纳米结构平台，能够高效形成3D多细胞癌症球体，并利用电化学信号作为球体活性的指标，实时、非破坏性地评估姜黄素等抗癌化合物在脑肿瘤共培养模型中的效果，其检测灵敏度甚至高于传统的比色法[46]。这些技术与实时发光监测相辅相成，共同推动了活细胞分析向动态化、集成化和信息多元化的方向发展，为药物研发、毒性测试和基础生物学研究提供了更强大的工具。5. 数据解读、标准化与计算模型整合5.1 检测方法的比较、选择与标准化 选择细胞活性检测方法是一个需要综合权衡多种参数的复杂过程。研究表明，没有任何一种方法是完美的，其选择需综合考虑成本、速度、复杂性、检测目标以及细胞模型等多种因素[47]。例如，在评估念珠菌的抗菌疗效时，代谢活性检测（如XTT和刃天青法）和菌落形成单位计数等方法各有其适用场景[48]。对于三维细胞培养体系，传统的二维检测方法可能不再适用，需要专门针对三维结构（如肿瘤球体）进行优化的方案，例如使用WST-8（CCK-8）试剂盒[49]。此外，细胞模型本身也极大地影响着方法的选择。传统的二维培养无法模拟体内三维微环境的复杂性，因此在药物开发和毒性研究中，需要能够更准确反映细胞生理状态的三维培养体系及其相应的活性检测技术[1]。为了更可靠地报告受试治疗剂的有效剂量，建议同时使用至少两种基于不同原理的检测方法，例如一种测量代谢健康，另一种检查膜通透性，以相互补充和验证[47]。这种策略在评估棘阿米巴原虫的细胞毒性时得到了体现，研究发现，基于水溶性四唑盐-甲臜或NanoLuc®荧光素酶底物的检测方法，相较于乳酸脱氢酶（LDH）释放法，能更准确地量化其对人类角膜上皮细胞的细胞毒性效应[50]。在建立适用于特定目的的检测方法时，需要采用实验与计算相结合的方法。一项研究通过系统性地使用热损伤Jurkat细胞，并在处理后使用膜完整性、代谢功能和凋亡等多种方法测量细胞活性，再与基于Gompertz分布的长期增殖动力学模型相关联，从而对这些检测方法作为细胞增殖预测指标进行排序，并为满足特定增殖反应要求设定细胞活性标准[51]。这种方法学框架为开发“适合目的”的细胞活性检测提供了系统性的途径。5.2 机制性计算模型在解读细胞异质性中的作用 细胞活性检测数据反映了群体中累积的分裂和死亡事件，蕴含着显著的细胞异质性[52]。然而，直接用机制性计算模型解释这些数据存在困难，因为直接进行模型与数据的比较往往模糊不清[52]。新开发的算法能够追踪机制详细的单细胞谱系中的模拟分裂和死亡事件，从而实现模型与数据的直接比较，并提示细胞间药物反应变异性的原因[52]。应用该模型模拟靶向抗癌药物（如曲美替尼、帕博西尼）的剂量反应，并与实验数据比较，不一致之处揭示了可能被高估的生物学机制以及影响药物反应的关键决定因素[52]。例如，模拟结果与曲美替尼（MEK抑制剂）强生长抑制的数据一致，但与帕博西尼（CDK4/6抑制剂）的数据不一致，这提示驱动细胞周期的CDK4/6的重要性可能被高估了[52]。此外，模型与数据的不一致还表明，基础信号与配体诱导信号之间的平衡是受体抑制剂反应的关键决定因素[52]。模拟结果还显示，在对照和药物治疗条件下都存在快速分裂和缓慢分裂的细胞亚群，而药物处理前母细胞中影响ERK通路活性的变异与快速分裂表型和曲美替尼耐药相关[52]。另一项研究也报道了类似的发现，通过机制建模和计算机谱系追踪，揭示了细胞异质性在药物反应中的决定因素，为将机制建模应用于大规模细胞活性数据集奠定了基础[9]。这项工作不仅有助于理解药物反应的变异性，也为更精准地预测药物疗效和耐药性提供了新的计算工具。5.3 基于细胞区室和生化元素的分类新框架 随着对多种细胞毒性通路机制认识的深入，有必要对用于评估细胞活性和细胞死亡的多种检测方法进行修订分类[24]。新的分类框架基于所涉及的细胞区室和/或生化元素，重点关注过程的机制和基本方面[24]。这些检测方法建立在不同的细胞功能之上，包括代谢活性、酶活性、细胞膜通透性和完整性、ATP含量、细胞粘附、还原当量、染料摄入或排除、组成型蛋白酶活性、集落形成、DNA断裂和核分裂等[24]。这种基于机制的分类有助于研究者根据具体的生物学问题和检测目标，更系统、更合理地选择和应用最合适的检测方法组合。例如，评估代谢活性的方法（如MTT、刃天青法）通过测量细胞的还原能力来间接反映活性[53]。而直接评估细胞膜完整性的方法，如新开发的膜电位细胞活性检测法，通过测量细胞膜的电势来直接判断细胞的存活状态，为评估不同细胞死亡通路提供了有前景的工具[54]。对于三维生物打印构建体，评估细胞活性面临独特挑战，自动化共聚焦显微镜结合活/死染色能提供比传统手动计数或间接代谢检测（如alamarBlue）更准确、可重复的结果[55]。在三维水凝胶构建体中，不同商业化的活性检测方法（如CellTiter-Glo®、PrestoBlue™）的准确性因水凝胶配方而异，强调了结合显微成像进行验证的重要性[3]。此外，针对特定应用场景，如微流体三维组织培养，定量图像细胞活性检测技术通过使用细胞非渗透性核染料（如EthD-1和DAPI）顺序标记坏死细胞和总细胞核，实现了在微小尺度下的精确量化[5]。这种基于细胞区室和功能的分类框架，使得各种检测方法能够作为评估新化疗生物分子在药物开发过程中对细胞存活影响的直接、可靠、灵敏、可重复、经济高效且高通量的手段[24]。结论 细胞活性检测技术的发展历程，清晰地勾勒出从静态、群体平均到动态、单细胞及复杂生理环境模拟的演进路径。这一演变不仅是技术工具的革新，更是研究范式从“描述现象”向“解析机制”和“预测行为”的深刻转变。当前的技术格局呈现出多元化与互补性并存的特点，传统终点法因其成熟与简便，在标准化高通量筛选中仍不可或缺；而新兴的实时、无标记及单细胞技术，则为揭示细胞行为的时空动态与异质性提供了前所未有的窗口。 从专家视角审视，这一领域的发展核心在于如何平衡技术的“深度”与“广度”，以及“分辨率”与“通量”。例如，在复杂3D模型（如类器官）中应用活性检测，其挑战远非简单移植2D技术所能解决。它要求我们审慎权衡检测探针的渗透深度与生物相容性、成像技术的三维解析能力与光毒性、以及从复杂信号中提取特定生物学信息的算法可靠性。解决之道在于采取一种综合策略：将经过优化的特异性分子探针与先进光学成像（如光片显微镜、共聚焦深层成像）相结合，并辅以多参数（如代谢、膜完整性、凋亡标志物）同步检测，方能对3D构建体内的细胞状态做出更接近生理真实的评估。 新兴技术如电化学传感和激光干涉显微镜，其价值在于实现了对细胞过程的“无扰”观测，极大减少了因标记物引入可能带来的实验偏差。然而，这些技术的数据解读往往需要更专业的背景知识，且其标准化和普及仍面临挑战。因此，未来的一个重要方向是推动这些高信息量技术的操作流程和数据输出标准化，使其能更广泛地整合到常规研究流程中。 尤为关键的是，对细胞活性数据的解读必须超越简单的“活性/非活性”二分法，深入细胞群体的异质性层面。单细胞分析技术已揭示，即使在同一处理下，细胞反应也存在显著差异，这直接关系到药物疗效与耐药性。通过整合机制性计算模型（如基于通路的药效动力学模型）与高通量实验数据，我们能够量化这种异质性，并追溯其背后的分子网络扰动根源。这种“干湿结合”的研究模式，是连接细胞表型观测与深层生物学机制、最终实现精准预测药物反应的关键桥梁。 展望未来，细胞活性检测体系的理想形态将是一个高度集成化、智能化和信息化的平台。它需要：1）开发具有智能响应特性、能穿透复杂基质的下一代探针；2）推动无标记、多参数实时监测技术从实验室走向标准化应用；3）深度融合计算生物学，利用人工智能和机器学习构建能够模拟和预测细胞在扰动下动态反应的“数字孪生”模型。最终目标是构建一个灵活、精准且信息维度丰富的评估体系，使其不仅能更真实地反映体外模型的生理与病理状态，更能为药物开发管线提供更具预测价值的临床前数据，加速从基础研究发现向转化医学与个性化医疗应用的迈进。参考文献 [1] Congress Z, Brovold M, Soker S. 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Peer-reviewed preclinical melanoma research finds Provectus’s synthetic small molecule can both stimulate anti-tumor immunity and suppress peripheral immune cells at the same dose. A recently published peer-reviewed medical journal article in Molecular Therapy: Oncology by OncoThAI researchers at the University of Lille in France shows that RBS can be immunostimulatory in tumor cells and immunosuppressive in peripheral immune cells at the same dose. Low-dose RBS combined with photodynamic activation in melanoma cells avoids the immunosuppressive effect observed at high dose while achieving equivalent tumor cell kill at a fraction of the dose. The preclinical study was conducted independently of Provectus, illustrating the value of externally generated validation that the Company’s recently launched Veripure ™ open science research program is designed to produce. KNOXVILLE, Tenn., July 07, 2026 (GLOBE NEWSWIRE) -- Provectus Biopharmaceuticals, Inc. (“Provectus” or the “Company”) (OTCQB: PVCT), a clinical-stage biotechnology company developing pharmaceutical-grade rose bengal sodium (“RBS”) active pharmaceutical ingredient (“API”) as a platform therapeutic across oncology, dermatology, ophthalmology, hematology, infectious disease, wound healing, tissue repair, neurodegeneration, and other diseases, today highlighted the publication of a peer-reviewed medical journal article describing a key property of RBS. The Company’s proprietary synthetic small molecule can produce opposing immune effects across distinct cell populations at the same dose. Independent, unsolicited validation of the multiple mechanisms of action of drug product candidate PV-10, formulated from RBS API, is among the most credible evidence of RBS’s platform capabilities. It reflects science robust enough to withstand investigation the Company did not commission or control. The paper, titled “ Innovative Photodynamic Therapy Using Rose Bengal for the Treatment of Human Melanoma ,” was published in Molecular Therapy: Oncology (Vol. 34, June 2026), published by Elsevier on behalf of the American Society of Gene and Cell Therapy. The study was conducted by French researchers at OncoThAI – Laser Assisted Therapies and Immunotherapies for Oncology at the University of Lille (“OncoThAI”) using PV-10 supplied by Provectus. OncoThAI aims to offer photodynamic therapy (“PDT”) for cancers with no remaining treatment options. Provectus recently announced the launch of its Veripure open science research program to make PV-10 freely available to qualified researchers worldwide. Researchers interested in studying RBS for therapeutic applications through the Veripure program are invited to contact Provectus directly at veripure@pvct.com . Key Finding: Opposing Immune Activity at the Same Dose Because of specific interest in using PDT to treat cancer, OncoThAI researchers investigated RBS as a photosensitizer in human metastatic melanoma. They combined low-dose RBS with green light to evaluate PDT effects in different melanoma cell lines. The authors asked whether RBS’s known photosensitizing properties could be harnessed to reduce therapeutic dose while preserving or enhancing antitumor activity. The Company considers the central finding of the paper to be this: at the same absolute dose, RBS produced opposing mechanistic effects in two distinct cell populations. In tumor cells, high-dose RBS killed melanoma cells predominantly through necrosis. Per the broader RBS literature the authors cite, this form of cell death releases damage-associated molecular patterns (“DAMPs”) — including high mobility group box 1 (HMGB1), calreticulin, and heat shock protein 90 (HSP90) — that innate immune sensors recognize, activating dendritic cells and triggering downstream adaptive responses against tumor antigens. In peripheral immune cells, evaluated in parallel experiments, the same RBS dose significantly suppressed proliferation of peripheral blood mononuclear cells (“PBMCs”) in non-activated and PHA-activated conditions. Put simply: High-dose RBS was immunostimulatory toward the tumor it was killing and immunosuppressive toward the peripheral immune cells positioned to respond to that killing. These are two concurrently opposing mechanistic effects by the same molecule at the same dose. RBS’s capacity to express different, even opposing, biological activities — sometimes at once, sometimes over time — depending on dose and cellular context is the same underlying property that gives the molecule its platform breadth across its full range of indications. The OncoThAI paper demonstrates that this same context-dependence can be directed, not merely observed. The French researchers showed that PDT is a treatment strategy that can direct this dose-dependent biology, concentrating RBS’s tumor-killing activity locally while keeping peripheral immune cells below the concentration threshold associated with suppression. Combined with green light, PDT-RBS was far more potent than RBS alone: 121 times more effective at reducing melanoma cell viability in HBL cells and 40 times more effective in LND cells. To reach 80% cell death, this cut the required RBS dose roughly 15-fold in HBL cells (492 µM to 32 µM) and 14-fold in LND cells (1,043 µM to 74 µM). At the lower dose, RBS avoided suppressing PBMC proliferation. Conditioned media from PDT-RBS–treated melanoma cells significantly increased proliferation of activated PBMCs at 72 and 120 hours. This response was consistent with the release of immunostimulatory soluble factors and the DAMP-driven immune activation cascade, without the immunosuppressive effect observed at high dose. Dominic Rodrigues, President and Vice Chairman of the Board of Provectus, noted, “The OncoThAI paper does something that has not been shown before in RBS literature. The data demonstrate that RBS can express multiple biological activities — some concurrent, some sequential — whose relative dominance depends on molecular quantity and cellular context. OncoThAI’s data, arrived at independently, show that dose and delivery strategy can direct which of RBS’s activities dominate in a given tissue compartment. That insight deepens our understanding of RBS’s therapeutic pro its full indication spectrum.” Consistent with Veripure’s Open Science Philosophy This study was conducted in a non-sponsored setting using PV-10 supplied to OncoThAI under the practice of unconstrained academic research access that the Company has maintained for years and has since formalized under its Veripure open science program. Researchers receive PV-10 drug product candidate, formulated from pharmaceutical-grade RBS API, without restrictions on research direction, design, or findings, and without publication controls, strict data-sharing requirements, or indication limits. Mr. Rodrigues added, “We did not commission this study, design its hypotheses, or direct its conclusions. OncoThAI asked their own question — whether photodynamic activation could optimize RBS’s dose-response profile — and arrived at a finding that meaningfully advances our understanding of the molecule’s multi-mechanistic behavior. That is precisely the kind of validation Veripure is designed to produce: independent researchers, working from their own hypotheses, generating findings of real scientific consequence using the same pharmaceutical-grade material our clinical programs depend on.” About Provectus Provectus Biopharmaceuticals, Inc. is a clinical-stage biotechnology company developing a pipeline of immunotherapy medicines based on rose bengal sodium, a first-in-class synthetic small molecule from the halogenated xanthene family. The Company’s clinical programs span oncology, dermatology, and ophthalmology, with additional proof-of-concept and early discovery programs in hematology, wound healing, infectious diseases, tissue repair, and neurodegeneration. For more information, visit . Forward Looking Statements The information in this press release may include “forward-looking statements,” within the meaning of the Private Securities Litigation Reform Act of 1995, relating to the business of Provectus and its affiliates, which are based on currently available information and current assumptions, expectations, and projections about future events and are subject to a variety of risks and uncertainties and other factors that could cause actual events or results to differ materially from those projected in the forward-looking statements. Forward-looking statements are often, but not always, identified by the use of words such as “aim,” “likely,” “outlook,” “seek,” “anticipate,” “budget,” “plan,” “continue,” “estimate,” “expect,” “forecast,” “may,” “will,” “would,” “project,” “projection,” “predict,” “potential,” “targeting,” “intend,” “can,” “could,” “might,” “should,” “believe,” and similar words suggesting future outcomes or statements regarding an outlook. The safety and efficacy of the agents and/or uses under investigation have not been established. There is no guarantee that the agents will receive health authority approval or become commercially available in any country for the uses being investigated or that such agents as products will achieve any particular revenue levels. Due to the risks, uncertainties, and assumptions inherent in forward-looking statements, readers should not place undue reliance on these forward-looking statements. The forward-looking statements contained in this press release are made as of the date hereof or as of the date specifically specified herein, and Provectus undertakes no obligation to update or revise any forward-looking statements, whether as a result of new information, future events or otherwise, except in accordance with applicable securities laws. The forward-looking statements are expressly qualified by this cautionary statement. Risks, uncertainties, and assumptions include those discussed in the Company’s filings with the Securities and Exchange Commission, including those described in Item 1A of the Company’s Annual Report on Form 10-K for the period ended December 31, 2025 and Quarterly Report on Form 10-Q for the period ended March 31, 2026 . Contacts: Provectus Biopharmaceuticals, Inc. Heather Raines, CPA Chief Financial Officer hraines@pvct.com (866) 594-5999 Investor Relations & Media Susan Xu sxu@allianceadvisors.com (778) 323-0959