体外培养杂交瘤细胞生产人用鼠源单克隆抗体

<正>目前,人用鼠源单抗的生产方法一般分为体内法和体外法2种。体内法即腹水法。尽管腹水中抗体浓度比较高（2～10mg/ml）,但由于所用的BALB/c小鼠必须达到SPF级,繁殖、饲养BALB/c小鼠及生产腹水、纯化抗体的厂房必须符合GMP要求,WHO及我国对体内法生产的人用鼠源单克隆抗体的质检项目繁多,要求严格,因此限制了体内法在人用鼠源单抗生产领域的应用。由于体外法（杂交瘤细胞体外培养法）的产品纯度高,可以避免鼠类病毒的污染,简化质检项目,操作具有可控制性,适用于大规模工业生产,     

抗人A血型杂交瘤细胞高密度培养条件的研究

抗人 A 血型杂交瘤15B_4细胞在 CelliGen 细胞培养罐中进行连续灌注培养,细胞密度达1.6×10~7/ml。每天灌注新培养液量由1000ml 增到2300ml,第14天后又逐日减少至1000ml,死细胞数随着转速的增加和灌注量的减少而增多。血凝效价呈梯度上升,滴度达到2~(14),相当于腹水效价。培养6天 IgM 产量为0.2克/升/天,10～18天波动在1.45～1.8克/升/天。15B_4细胞代谢与葡萄糖、乳酸有关,与氨无关。CelliGen 自控效果较好,适用于杂交瘤细胞的高密度培养。搅拌速度在120～150r/min 对细胞产生的剪切力有明显地破坏作用。溶氧控制系统有待改进。     

牛布氏杆菌抗独特型杂交瘤细胞的悬浮培养研究

牛布氏杆菌抗独特型杂交瘤细胞F9株能稳定地分泌高效价的抗独特型抗体,其Ig类型为IgG2a,并具有抗原“内影象”和良好的免疫原性。该细胞培养在Techne－T104型1．5L细胞培养装置中,最适连续培养条件是pH7．1～7．5;36．5±0．5℃,搅拌速度35～45r／min。在连续悬浮培养时,细胞密度维持在3．2×105～8．2×105／ml,单克隆抗独特型抗体浓度可达1．04mg／ml,培养上清ELISA捕获法效价达1：160。     

简易的杂交瘤细胞载体培养法

在1．0L的搅拌培养瓶内安装一个可转动的不锈钢篮子，内充填8．0g的圆片状聚脂载体Fibra－Cel，杂交瘤细胞r－69B培养在此搅拌瓶中，转动速度为100r／min，结果53.5％细胞固滞在聚脂载体内，细胞浓度和产生抗人r－IFN的单克隆抗体IgG量为常规法的2倍左右。该设备简单，很适用于普通实验室培养杂交瘤细胞，也可以用于培养其他各种类型的人和动物细胞。     

杂交瘤细胞流加培养中葡萄糖和谷氨酰胺的代谢控制

对底物限制的流加培养中杂交瘤细胞HB 58的生长、代谢和单抗生成进行了研究。葡萄糖或 谷氨酰胺限制引起比生长速率下降。葡萄糖限制时,乳酸生成减少,细胞对葡萄糖的得率增 加,乳酸对葡萄糖的得率降低,说明葡萄糖更多地参与三羧酸循环。谷氨酰胺限制时,氨和丙氨酸生成减少,细胞和氨对谷氨酰胺的得率增加,丙氨酸对谷氨酰胺的得率减小,说明谷氨酰胺更多地参与脱氢反应,其有效利用率得到提高。     

无血清培养中杂交瘤细胞的能量代谢特性

在 10L生物反应器中 ,研究杂交瘤细胞C5 0在无血清培养条件下生长和能量代谢的特性。结果表明 ,细胞的比耗氧速率、ATP比生成速率和比生长速率有着相似的过程曲线 ,ATP比生成速率与比生长速率呈线性相关 ,反映了细胞能量代谢与生长之间的密切关系。同时发现 ,耗氧速率 (OUR)能够及时、敏感地反映细胞的代谢活力 ,预示细胞的生长变化。据此提出了利用OUR控制补料速率的初步设想。     

抗rhTNFα杂交瘤细胞的体外培养及抗体分泌的研究

采用非CO2依赖培养基（CO2-IM）在无CO2、37℃条件下培养抗rhTNFα杂交瘤细胞，培养1周PH无显著变化，细胞生长趋势和抗体分泌显著超过DMEM。将CO2－IM与SFPF－HM混合后添加ITSE可以培养抗rhTNF杂交瘤细胞，其细胞生长速率、密度、活力和抗体产量均达到或超过含血清培养。     

应用转瓶大规模培养杂交瘤细胞生产ABO血型单克隆抗体的工艺

目的建立用转瓶悬浮培养杂交瘤细胞生产ＡＢＯ血型抗原单抗的工艺。方法 在无ＣＯ２条件下, 应从转瓶培养分泌抗人Ａ和Ｂ血型抗原单抗的杂交瘤细胞。结果在适当的ｐＨ、转速和起始密度的条件下,细胞 的生长密度和单抗效价均超过静态培养水平。结论该工艺投资少,操作简便,运行稳定,适于规模化生产。     

应用篮式生物反应器培养1A_5杂交瘤细胞及单克隆抗体分泌

目的 为了提高体外培养的杂交瘤细胞单克隆抗体的合成分泌,并为治疗型人源化单克隆抗体生 产摸索条件。方法 应用５Ｌ篮式及非篮式生物反应器培养１Ａ５杂交瘤细胞,以分泌干扰素单克隆抗体,从中对 １Ａ５细胞的接种浓度、反应动态、培养方式等进行研究。结果 细胞最适的接种浓度为２．０×１０５／ｍｌ左右;同非篮式 培养相比,在篮式培养过程中,通过葡萄糖消耗率的监测,及时地调节培养条件及换液时间,延长了培养周期,增加 了每罐收液批次,并且每次是全量换液,最高收液效价为１：４０９６。结论（１）１Ａ５ 细胞分泌干扰素单抗属于负生长 耦连型,葡萄糖消耗率与细胞的生长呈线性关系,可以利用糖消耗率的变化判断篮式生物反应器中细胞生长状况; （２）篮式生物反应器载体培养优于非篮式生物反应器悬浮培养;（３）适当地增加培养基中葡萄糖的量,可以提高干 扰素单抗的合成分泌。     

小鼠-Vero细胞法测定白喉类毒素抗体试验条件的优化

目的优化小鼠-Vero细胞法测定白喉类毒素抗体的试验条件。方法用含吸附白喉类毒素的制品免疫NIH小鼠,35d后眼眶采血,分离血清,对用小鼠-Vero细胞法测定白喉类毒素抗体的试验条件进行优化。结果确定了小鼠-Vero细胞法测定白喉类毒素抗体的最佳试验条件。结论优化试验条件的小鼠-Vero细胞测定白喉类毒素抗体的方法,能客观准确地评价白喉类毒素免疫保护效力,且具有特异、灵敏、重复性好的特点。     

Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture,NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production

In this study, endophytic bacteria belonging to the Bacillus genus were isolated from in vitro bulblets of Leucojum aestivum and their ability to produce Amaryllidaceae alkaloids was studied. Proton Nuclear Magnetic Resonance (¹H NMR)-based metabolomics combined with multivariate data analysis was chosen to compare the metabolism of this plant (in vivo bulbs, in vitro bulblets) with those of the endophytic bacteria community. Primary metabolites were quantified by quantitative ¹H NMR (qNMR) method. The results showed that tyrosine, one precursor of the Amaryllidaceae alkaloid biosynthesis pathway, was higher in endophytic extract compared to plant extract. In total, 22 compounds were identified including five molecules common to plant and endophyte extracts (tyrosine, isoleucine, valine, fatty acids and tyramine). In addition, endophytic extracts were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) for the identification of compounds in very low concentrations. Five Amaryllidaceae alkaloids were detected in the extracts of endophytic bacteria. Lycorine, previously detected by ¹H NMR, was confirmed with LC-MS analysis. Tazettine, pseudolycorine, acetylpseudolycorine, 1,2-dihydro-chlidanthine were also identified by LC-MS using the positive ionization mode or by GC-MS. In addition, 11 primary metabolites were identified in the endophytic extracts such as tyramine, which was obtained by decarboxylation of tyrosine. Thus, Bacillus sp. isolated from L. aestivum bulblets synthesized some primary and specialized metabolites in common with the L. aestivum plant. These endophytic bacteria are an interesting new approach for producing the Amaryllidaceae alkaloid such as lycorine.     

稀土元素对水母雪莲细胞生长及黄酮类化合物合成的影响

研究了稀土元素钕(Nd3+)、铈(Ce3+)、镧(La3+)和混合稀土(MRE)对摇瓶液体培养的水母雪莲细胞生长及黄酮类化合物合成的影响. 发现Ce3+和La3+及混合稀土可促进雪莲细胞的生长及黄酮类化合物的合成,其中以Ce3+效果最佳. 当初始浓度为0.025 mmol/L的Ce3+添加到改良的MS培养基中时,细胞生物量和黄酮类化合物产量最高,分别可达17.7 g/L及942 mg/L,分别是不添加稀土元素的对照实验的134.4%和166.7%;同时发现在培养基中不含外源激素6-BA的条件下,合适浓度的Ce3+可替代6-BA对雪莲细胞生长及黄酮类化合物合成的促进作用,而在培养基中不含NAA时,Ce3+不能替代NAA对雪莲细胞生长及黄酮类化合物合成的促进作用.     

Release Kinetics and In Vitro Characterization of Sodium Percarbonate and Calcium Peroxide to Oxygenate Bioprinted Tissue Models

Oxygen-generating materials have been used in several tissue engineering applications; however, their application as in situ oxygen supply within bioprinted constructs has not been deeply studied. In this study, two oxygen-generating materials, sodium percarbonate (SPO) and calcium peroxide (CPO), were studied for their oxygen release kinetics under a 0.1% O₂ condition. In addition, a novel cell-culture-insert setup was used to evaluate the effects of SPO and CPO on the viability of skeletal muscle cells under the same hypoxic condition. Results showed that SPO had a burst oxygen release, while CPO had a more stable oxygen release than SPO. Both SPO and CPO reduced cell viability when used alone. The addition of catalase in SPO and CPO increased the oxygen release rate, as well as improving the viability of skeletal muscle cells; however, CPO still showed cytotoxicity with catalase. Additionally, the utilization of 1 mg/mL SPO and 20 U catalase in a hydrogel for bioprinting significantly enhanced the cell viability under the hypoxic condition. Moreover, bioprinted muscle constructs could further differentiate into elongated myotubes when transferring back to the normoxic condition. This work provides an excellent in vitro model to test oxygen-generating materials and further discover their applications in bioprinting, where they represent promising avenues to overcome the challenge of oxygen shortage in bioprinted constructs before their complete vascularization.     

Applications of Biomaterials in 3D Cell Culture and Contributions of 3D Cell Culture to Drug Development and Basic Biomedical Research

The process of evaluating the efficacy and toxicity of drugs is important in the production of new drugs to treat diseases. Testing in humans is the most accurate method, but there are technical and ethical limitations. To overcome these limitations, various models have been developed in which responses to various external stimuli can be observed to help guide future trials. In particular, three-dimensional (3D) cell culture has a great advantage in simulating the physical and biological functions of tissues in the human body. This article reviews the biomaterials currently used to improve cellular functions in 3D culture and the contributions of 3D culture to cancer research, stem cell culture and drug and toxicity screening.     

From Primary MSC Culture of Adipose Tissue to Immortalized Cell Line Producing Cytokines for Potential Use in Regenerative Medicine Therapy or Immunotherapy

For twenty-five years, attempts have been made to use MSCs in the treatment of various diseases due to their regenerative and immunomodulatory properties. However, the results are not satisfactory. Assuming that MSCs can be replaced in some therapies by the active factors they produce, the immortalized MSCs line was established from human adipose tissue (HATMSC1) to produce conditioned media and test its regenerative potential in vitro in terms of possible clinical application. The production of biologically active factors by primary MSCs was lower compared to the HATMSC1 cell line and several factors were produced only by the cell line. It has been shown that an HATMSC1-conditioned medium increases the proliferation of various cell types, augments the adhesion of cells and improves endothelial cell function. It was found that hypoxia during culture resulted in an augmentation in the pro-angiogenic factors production, such as VEGF, IL-8, Angiogenin and MCP-1. The immunomodulatory factors caused an increase in the production of GM-CSF, IL-5, IL-6, MCP-1, RANTES and IL-8. These data suggest that these factors, produced under different culture conditions, could be used for different medical conditions, such as in regenerative medicine, when an increased concentration of pro-angiogenic factors may be beneficial, or in inflammatory diseases with conditioned media with a high concentration of immunomodulatory factors.     

Degradation Products of Tryptophan in Cell Culture Media: Contribution to Color and Toxicity

Biomanufacturing processes may be optimized by storing cell culture media at room temperature, but this is currently limited by their instability and change in color upon long-term storage. This study demonstrates that one of the critical contributing factors toward media browning is tryptophan. LC-MS technology was utilized to identify tryptophan degradation products, which are likely formed primarily from oxidation reactions. Several of the identified compounds were shown to contribute significantly to color in solutions but also to exhibit toxicity against CHO cells. A cell-culture-compatible antioxidant, a-ketoglutaric acid, was found to be an efficient cell culture media additive for stabilizing components against degradation, inhibiting the browning of media formulations, and decreasing ammonia production, thus providing a viable method for developing room-temperature stable cell culture media.     

In Vitro Effects of Selective COX and LOX Inhibitors and Their Combinations with Antineoplastic Drugs in the Mouse Melanoma Cell Line B16F10

The constitutive expression or overactivation of cyclooxygenase (COX) and lipoxygenase (LOX) enzymes results in aberrant metabolism of arachidonic acid and poor prognosis in melanoma. Our aim is to compare the in vitro effects of selective COX-1 (acetylsalicylic acid), COX-2 (meloxicam), 5-LOX (MK-886 and AA-861), 12-LOX (baicalein) and 15-LOX (PD-146176) inhibition in terms of proliferation (SRB assay), mitochondrial viability (MTT assay), caspase 3-7 activity (chemiluminescent assay), 2D antimigratory (scratch assay) and synthesis of eicosanoids (EIA) in the B16F10 cell line (single treatments). We also explore their combinatorial pharmacological space with dacarbazine and temozolomide (median effect method). Overall, our results with single treatments show a superior cytotoxic efficacy of selective LOX inhibitors over selective COX inhibitors against B16F10 cells. PD-146176 caused the strongest antiproliferation effect which was accompanied by cell cycle arrest in G₁ phase and an >50-fold increase in caspases 3/7 activity. When the selected inhibitors are combined with the antineoplastic drugs, only meloxicam provides clear synergy, with LOX inhibitors mostly antagonizing. These apparent contradictions between single and combination treatments, together with some paradoxical effects observed in the biosynthesis of eicosanoids after FLAP inhibition in short term incubations, warrant further mechanistical in vitro and in vivo scrutiny.     

In Vitro Tumor Cell-Binding Assay to Select High-Binding Antibody and Predict Therapy Response for Personalized 64Cu-Intraperitoneal Radioimmunotherapy against Peritoneal Dissemination of Pancreatic Cancer: A Feasibility Study

Peritoneal dissemination of pancreatic cancer has a poor prognosis. We have reported that intraperitoneal radioimmunotherapy using a ⁶⁴Cu-labeled antibody (⁶⁴Cu-ipRIT) is a promising adjuvant therapy option to prevent this complication. To achieve personalized ⁶⁴Cu-ipRIT, we developed a new in vitro tumor cell-binding assay (⁶⁴Cu-TuBA) system with a panel containing nine candidate ⁶⁴Cu-labeled antibodies targeting seven antigens (EGFR, HER2, HER3, TfR, EpCAM, LAT1, and CD98), which are reportedly overexpressed in patients with pancreatic cancer. We investigated the feasibility of ⁶⁴Cu-TuBA to select the highest-binding antibody for individual cancer cell lines and predict the treatment response in vivo for ⁶⁴Cu-ipRIT. ⁶⁴Cu-TuBA was performed using six human pancreatic cancer cell lines. For three cell lines, an in vivo treatment study was performed with ⁶⁴Cu-ipRIT using high-, middle-, or low-binding antibodies in each peritoneal dissemination mouse model. The high-binding antibodies significantly prolonged survival in each mouse model, while low-and middle-binding antibodies were ineffective. There was a correlation between in vitro cell binding and in vivo therapeutic efficacy. Our findings suggest that ⁶⁴Cu-TuBA can be used for patient selection to enable personalized ⁶⁴Cu-ipRIT. Tumor cells isolated from surgically resected tumor tissues would be suitable for analysis with the ⁶⁴Cu-TuBA system in future clinical studies.