Earticle

The aim of the present study is to evaluate effects of Spheroid 9000NS applied to 10 elderly patients with Chronic Low Back Pain. The effects were measured and analyzed by means of MPQWL, VRS, VAS-I and VAS-U after 4, 8 and 12 weeks, respectively, during the 12-week application of the Spheroid 9000NS. From the findings of the measurement and analysis, following conclusion could be drawn: Significant reductions in MPQWL were confirmed at each period of measurement since the use of Spheroid 9000NS(p<.001). Significant reductions were also revealed in VRS at each period of the measurement(p<.001). Significant reductions were also revealed in VAS-I at each period of the measurement(p<.001). The same reductions were found as well in VAS-U 4, 8 and 12 weeks after the application of the Spheroid 9000NS(p<.001). In sum, it may be concluded from the outcomes that the use of Spheroid 9000NS may help reduce drastically Chronic Low Back Pain, mostly effective to remove them speedily and safely. Patients, rehabilitation therapists and health experts are, therefore, recommended to employ the products as safe and effective means to control pains. Continued researches and experiments should further be conducted to collect more data from the larger population of patients with particular diseases in clinical situation, though.

Current studies have revealed the capacity of mesenchymal stem cells (MSCs) in term of immunomodulatory properties, and this distinct potential is downgraded according to the disease duration of patients-derived MSCs. In order to enhance the immunomodulatory and anti-tumorigenic properties of the rheumatoid arthritis (RA) joints-derived MSCs, we aggregate synovial fluid-derived MSCs from RA joints (RA-hMSCs) into 3D-spheroids by the use of hanging drop culture method. Cells were isolated from synovial fluids of RA joints with longstanding active status over 13 years. For aggregation of RA-hMSCs into 3D-spheroids, cells were plated in hanging drops in 30 μL of advanced DMEM (ADMEM) containing 25,000-30,000 cells/ drop and cultured for 48 h. To analyze the comparative immunomodulatory effects of 3D-spheroid and 2D monolayer cultured RA-hMSCs and then cells were cultured in ADMEM supplemented with 20% of synovial fluids of RA patients for 48 h and were evaluated by qRT-PCR for their expression of mRNA levels of inflammatory and antiinflammatory markers. Cellular aggregation of RA-hMSCs was observed and cells were aggregate into a single sphere. Following treatment of RA patient’s synovial fluids into the RA-hMSCs, spheroids formed RA-hMSCs showed significantly (p < 0.05) higher expression of TNFα stimulated gene/protein 6 (TSG-6) than the monolayer cultured RAhMSCs. Therefore, the 3D-spheroid culture methods of RA-hMSCs were more effective than 2D monolayer cultures in suppressing inflammatory response treated with 20% of RA-synovial fluids by expression of TNFα (TSG-6) according to the immune response and enhanced secretion of inflammatory factors.

Primary hepatocytes (PH) are considered as “gold standard” for drug screening because of its ability to express the entire set of drug metabolizing enzymes and transporters. Hepatocytes culturing and maintaining their hepatocyte fate in vitro is one of the major issue from last decade. The main problem in hepatocytes in vitro culturing is that, these cells rapidly loss their hepatic morphology and liver specific function in culture condition. In the present study, we isolated rat PH and cultured in monolayer (2D) as well as spheroid (3D) culture system. The 2D cultured PH hepatocyte showed an elongated hepatocyte morphology while, 3D cultured PH showed spheroid morphology with gradual decrease in diameter up to 7 days. After 7 days of in vitro culture, these cells were analyzed for the expression of hepatic markers (Alb, Tf, Afp) and apoptotic markers (Bax, Bcl2). Furthermore, PH in both culture systems was induced with two inducers i.e. 3-methylcholanthrene (3-MC, Cyp1a specific inducer) and dexamethasone (Cyp3a specific inducer) for 48 and 72 hours, respectively. The mRNA level of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PH, respectively. After 7 days in vitro culture, PH showed dramatic down regulation of hepatic markers in both culture systems. Furthermore, expression of apoptotic markers was higher in 2D cultured PH as compared to 3D. Cyp1a and Cyp3a mRNA level showed higher RNA content in 2D culture PH after 48 of induction. Therefore, we concluded that there was no significant difference found in two culture system and further studies are needed to find out the essential components for PH in vitro culture rather than culture system.

석기를 제작하는 것은 석기의 외형적 특징을 석재에 구현하기 위하여 격지를 떼어내는 과정이다. 석기 표면의 둔각은 여러면석기의 외형적 특징을 규정하는 중요한 요소로서 이것은 여러면석기의 둔각은 몸체를 둥글게 만들려고 하는 목적에서 구현된 필연적 결과이다. 석기의 표면에 둔각을 구현하는 방법은 다섯 가지로 정리할 수 있다. ① 타격면과 격지 끝이 위치하는 면의 연장선이 예각을 이루는 경우 망치의 궤적을 극단적으로 몸체에 근접한 타격을 하여 격지를 떼어낸다. ②몸체의 모서리가 직각을 이루는 경우 모서리의 끝을 타격하여 격지를 떼어낸다. ③ 타격면과 격지 끝이 위치하는 면의 연장선이 둔각을 이루는 경우는 격지를 뗄 수 없으므로 모서리를 부숴 둔각을 만든다. ④ 격지는 두꺼운 부분을 따라 떨어지는 성질을 이용하여 면과 면이 만나는 능선을 제거하여 둔각을 만든다. ⑤세면이 만나는 꼭짓점을 제거하여 둔각을 만든다. 둔각을 떼는 타격은 한 번의 타격으로 격지가 떨어지지 않으므로 둔각을 만드는 과정에서 발생하는 격지는 파손되는 경우가 많으며, 여러면석기 몸체에 남아있는 격지 자국도 상당한 교란이 되어 있다. 앞으로 여러면석기 제작 과정에서 발생한 격지에 대한 연구와 함께 제작과정에서 실패한 여러면석기 미완성품에 대한 연구를 진행한다면 여러면석기와 몸돌의 경계를 규명하는데 단초를 제공할 수 있을 것이다.

Making stone tools is the flaking process to express the external features of stone tools on the stone material. The obtuse angle on the surface of the stone tools is an essential factor defining the external features of the polyhedron, which is an inevitable outcome from achieving the purpose of making the body round. There are five methods of creating an obtuse angles on the surface of stone tools. ① If the extension line of the surface where the striking surface and the flaking end are located, forms an acute angle, strike the locus of hammer extremely close to the body and remove the flake. ② If the edge of the body is at a right angle, strike the end bit of the edge to flake it. ③ If the extension line of the surface where the striking surface and flaking end are located, forms an obtuse angle, break the edge to create an obtuse angle because it cannot be removed. ④ When flaking, make an obtuse angle by removing the ridge where the sides meet using the property of falling apart along the thick part. ⑤ Create an obtuse angle by removing the vertices where the three sides meet. When making obtuse angles, it requires more than one flaking step. So, the flakes are often damaged in the process of making an obtuse angle, and the scattered marks left on the body of the stone tools on many sides are also quite disturbing. More studies on the flakes generated by the manufacturing process of polyhedron and on the unfinished products of the polyhedron that failed in the manufacturing process shall provide a clue in classifying the boundary between the polyhedron and the core in the future.

실제영상으로 가상환경을 구축해서 사용자가 가상환경을 돌아다님으로써 보다 큰 몰입감과 현실감을 제공하는 영상기반 가상현실 기술은 최근 들어 웹 기반 가상현실시스템을 구축하기 위해서 많이 사용된다. 이 기술은 가상환경 구축에 있어 항해를 쉽게 하기 위한 한 방법으로 넓은 시각 영역(field of view)을 얻을 수 있는 wide-angle 렌즈를 흔히 사용한다. 어안렌즈(fish-eye lens)는 전형적인 넓은 시각 영역을 가진 렌즈로서, 매우 큰 radial distortion 을 가진다. 왜곡을 없앤 영상을 얻기 위해 본 논문에서는 구면기하(spherical geometry) 및 사영기하(projective geometry)를 사용하여 어안영상을 보정하는 non-metric기법을 제안한다. 제안한 이 방법은, 기존의 방법들 보다는 쉽고 빠른 속도로 왜곡을 보정할 수 있으므로 어안영상의 왜곡을 보정하는 하드웨어를 효율적으로 구현할 수 있다. 그리고 spheroid 를 이용해 좀 더 왜곡을 정확히 보정방법과 별도의 서보 모터 없이 pan/title 를 가능케 하는 시점이동에 따른 왜곡 보정 방법을 제시한다.

암조직의 내부에서 hypoxia와 glucose depletion 등의 microenvironmental stress를 받게 되면 necrosis가 유도되고, 실제로 암 조직 내부에서 necrotic core 형성이 관찰된다. Necrotic cells은 high mobility group box 1(HMGB1)를 extracellular space로 방출하는 것으로 알려져 있다. 방출된 HMGB1은 tumor-promoting cytokine으로 작용함으로써 tumor development 시 inflammation, metabolism 및 metastasis에 기여한다. 본 연구에서 non-invasive breast cancer cells MCF-7이 solid tumor의 in vitro model인 multicellular tumor spheroid (MTS) 배양을 통해 완전한 구형의 MTS를 형성하며 MTS가 성장함에 따라 inner region에 necrosis가 유도됨을 밝혔다. 또한 MCF-7 세포의 MTS 배양은 Snail 의존적으로 epithelial-mesenchymal transition (EMT)를 유도함을 관찰하였다. HMGB1의 cell surface receptors인 RAGE, TLR2, TLR4 발현이 MTS 배양에 의해 증가됨을 발견하였다. RAGE, TLR2, TLR4 를 knockdown한 결과 MTS 성장을 억제할 뿐만 아니라 MTS에 의해 증가되는 Snail 발현을 억제함을 밝혔다. 이는 MTS-induced Snail 발현이 RAGE/TLR2/TLR4의존적으로 조절되며 RAGE/TLR2/TLR4-Snail이 MTS 성장에 관여하는 것으로 보인다. 또한 Snail, RAGE, TLR2, TLR4 shRNA는 MTS 배양에 의해 유도되는 EMT를 억제함을 밝혔다. 실제 인간 암조직에서 정상조직에 비해 RAGE, TLR2, TLR4 유전자의 발현이 높음을 관찰하였다. 따라서 HMGB1이 RAGE/TLR2/4-Snail axis를 통해 MTS 배양에 따른 성장 및 EMT에 중요하게 작용할 것으로 예상된다.

As tumors develop, they encounter microenvironmental stress, such as hypoxia and glucose depletion, due to poor vascular function, thereby leading to necrosis, which is observed in solid tumors. Necrotic cells are known to release cellular cytoplasmic contents, such as high mobility group box 1 (HMGB1), into the extracellular space. The release of HMGB1, a proinflammatory and tumor-promoting cytokine, plays an important role in promoting inflammation and metabolism during tumor development. Recently, HMGB1 was shown to induce the epithelial-mesenchymal transition (EMT) and metastasis. However, the underlying mechanism of the HMGB1-induced EMT, invasion, and metastasis is unclear. In this study, we showed that noninvasive breast cancer cells MCF-7 formed tightly packed, rounded spheroids and that the cells in the inner regions of a multicellular tumor spheroid (MTS), an in vitro model of a solid tumor, led to necrosis due to an insufficient supply of O2 and glucose. In addition, after 7 d of MTS culture, the EMT was induced via the transcription factor Snail. We also showed that HMGB1 receptors, including RAGE, TLR2, and TLR4, were induced by MTS culture. RAGE, TLR2, and TLR4 shRNA inhibited MTS growth, supporting the idea that RAGE/TLR2/TLR4 play critical roles in MTS growth. They also prevented MTS culture-induced Snail expression, pointing to RAGE/TLR2/TLR4-dependent Snail expression. RAGE, TLR2, and TLR4 shRNA suppressed the MTS-induced EMT. In human cancer tissues, high levels of RAGE, TLR2, and TLR4 were detected. These findings demonstrated that the HMGB-RAGE/TLR2/TLR4-Snail axis played a crucial role in the growth of the MTS and MTS culture-induced EMT.

The olfactory system can detect many odorants with high sensitivity and selectivity based on the expression of nearly a thousand types of olfactory receptors (ORs) in olfactory receptor neurons (ORNs). These ORs have a dynamic odorant detection range and contribute to signal encoding processes in the olfactory bulb (OB). To harness the capabilities of the olfactory system and develop a biomimetic sensor, stable culture and maintenance of ORNs are required. However, in vitro monolayer culture models have several key limitations: i) short available period of cultured neurons, ii) low cultural efficiency, and iii) long-term storage challenges. This study aims to develop a technique: i) to support the spheroid culture of primary ORN precursors facilitating stable maintenance and long-term storage, and ii) to demonstrate the viability of ORN spheroid culture in developing an olfactory system mimetic bioelectronic nose. Recombinant protein (REP; TGPG[VGRGD(VGVPG)6]20WPC) was used to form the ORN spheroids. Spheroid formation enabled preservation of primary cultured ORNs without a significant decrease in viability or the expression of stemness markers for ten days. Physiological characteristics of the ORNs were verified by monitoring intracellular calcium concentration upon odorant mixture stimulation; response upon odorant stimulation were observed at least for ten days in these cultivated ORNs differentiated from spheroids. Coupling ORNs with multi electrode array (MEA) enabled the detection and discrimination of odorants by analyzing the electrical signal patterns generated following odorant stimulation. Taken together, the ORN spheroid culture process is a promising technique for the development of a bioelectronic nose and high-throughput odorant screening device.

Cell-based therapy is a promising approach in the field of regenerative medicine. As cells are formed into spheroids, their survival, functions, and engraftment in the transplanted site are significantly improved compared to single cell transplantation. To improve the therapeutic effect of cell spheroids even further, various biomaterials (e.g., nano- or microparticles, fibers, and hydrogels) have been developed for spheroid engineering. These biomaterials not only can control the overall spheroid formation (e.g., size, shape, aggregation speed, and degree of compaction), but also can regulate cell-to-cell and cell-to-matrix interactions in spheroids. Therefore, cell spheroids in synergy with biomaterials have recently emerged for cell-based regenerative therapy. Biomaterials-assisted spheroid engineering has been extensively studied for regeneration of bone or/and cartilage defects, critical limb ischemia, and myocardial infarction. Furthermore, it has been expanded to pancreas islets and hair follicle transplantation. This paper comprehensively reviews biomaterials-assisted spheroid engineering for regenerative therapy.

Most angiogenesis assays are performed using endothelial cells. However, blood vessels are composed of two cell types: endothelial cells and pericytes. Thus, co-culture of two vascular cells should be employed to evaluate angiogenic properties. Here, we developed an in vitro 3-dimensional angiogenesis assay system using spheroids formed by two human vascular precursors: endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs). ECFCs, MSCs, or ECFCs+MSCs were cultured to form spheroids. Sprout formation from each spheroid was observed for 24 h by real-time cell recorder. Sprout number and length were higher in ECFC+MSC spheroids than ECFC-only spheroids. No sprouts were observed in MSC-only spheroids. Sprout formation by ECFC spheroids was increased by treatment with vascular endothelial growth factor (VEGF) or combination of VEGF and fibroblast growth factor-2 (FGF-2). Interestingly, there was no further increase in sprout formation by ECFC+MSC spheroids in response to VEGF or VEGF+FGF-2, suggesting that MSCs stimulate sprout formation by ECFCs. Immuno-fluorescent labeling technique revealed that MSCs surrounded ECFC-mediated sprout structures. We tested vatalanib, VEGF inhibitor, using ECFC and ECFC+MSC spheroids. Vatalanib significantly inhibited sprout formation in both spheroids. Of note, the $IC_{50}$ of vatalanib in ECFC+MSC spheroids at 24 h was $4.0{\pm}0.40{\mu}M$, which are more correlated with the data of previous animal studies when compared with ECFC spheroids ($0.2{\pm}0.03{\mu}M$). These results suggest that ECFC+MSC spheroids generate physiologically relevant sprout structures composed of two types of vascular cells, and will be an effective pre-clinical in vitro assay model to evaluate pro- or anti-angiogenic property.

HeLa 세포의 spheroid를 배 양시켜 cis-platinum과 carboplatin으로 처리한 후 그 반응을 세포의 생존분획으로 분석하였다. 체외실험 model인 spheroid를 사용하여 platinum유사체의 약효와 방사선 감수성을 평가하고 약제에 대한 단층 세포와 spheroid의 감수성 차이를 세포-생존곡선에서 규명하기 위하여 본 실험을 시행하였다. Cis-platinum 농도-곡선에서 spheroid의 $Cq=3.4{\mu}M$이고 $Co=1.2{\mu}M$이었다. 이 것은 단층세포에 비하여 Co는 큰 변화가 없으나 Cq가 증가되어 cis-platinum이 저산소층 세포보다 활동적으로 분화하는 표면세포에 주로 작용하였으며, 반대로 carboplatin의 효과는 spheroid에 대한 $(Co=15.0{\mu}M)$ 감수성이 단층세포$(Co=32.5{\mu}M)$에 비하여 크게 증가되어, spheroid의 심층 세포에 주로 작용하였다. 방사선과 carboplation의 병용효과를 세포 생존분획이 0.01 수준에서 isobologram으로 분석한 결과 상호작용으로 supra-additive 효과를 나타내었다. 따라서, carboplatin은 cis-platinum에 비하여 신장과 위장에 대한 독성작용이 적고, 방사선과 병용함으로써 향후 더욱 효과적 인 종양 치료에 중요한 역할을 할 것으로 기대한다.

Multicellular tumor spheroids of HeLa cells have been grown in a static culture system. Samples of spheroids were exposed for 2 h to graded concentration of cis-platinum and its analogue, carboplatin, and then response assayed by survival of clonogenic cells. The purpose of present experiment is to clarify the effectiveness of these platinum compounds and to evaluate intrinsic radiosensitivity of cells using spheroids of HeLa cells as an experimental in vitro model. Variations of the drug sensitivity of monolayers as well as spheroids were also evaluated in cell-survival curves. In cis-platinum concentration-survival curve, there was a large shoulder extending as far as $Cq=3.4{\mu}M$, after which there was exponential decrease in survival curve having a Co Value of $1.2{\mu}M$ in spheroids. While the Co for the spheroids was essentially no significant change, but Cq value was larger than that of monolayers. This suggest that the effect of cis-platinum is greater En the monolayer with actively proliferaing cells than hypoxic one. In the carboplatin concentration-survival curves, the Co value of spheroids was $15.0{\mu}M$ and the ratio with the Co from monolayer cell $(32.5{\mu}M)$ was 0.40, thus indicating that the spheroids had a greater sensitivity to carboplatin than monolayers. Therefore, the effect of carboplatin is mainly on the deeper layers of spheroids acting as hypoxic cell sensitizer. The enhanced effect was obtained for monolayer cells using combined X-ray and carboplatin treatment 2 hours before irradiation. The result shown in isobologram analysis for the level of surviving fraction at 0.01 indicated that the effect of two agents was trusty supra-additive. From this experimental data, carboplatin has excited much recent interest as one of the most promising, since it is almost without nephrotoxicity and causes less gastrointestinal toxicity than cis-platinum. Interaction between carboplatin and radiation might play an important role for more effective local tumor control.

Mesenchymal stem cells (MSCs) offer significant therapeutic promise for various regenerative therapies. However, MSC-based therapy for injury exhibits low efficacy due to the pathological environment in target tissues and the differences between in vitro and in vivo conditions. To address this issue, we developed adipose-derived MSC spheroids as a novel delivery method to preserve the stem cell microenvironment. MSC spheroids were generated by suspension culture for 3 days, and their sizes increased in a time-dependent manner. After re-attachment of MSC spheroids to the plastic dish, their adhesion capacity and morphology were not altered. MSC spheroids showed enhanced production of hypoxia-induced angiogenic cytokines such as vascular endothelial growth factor (VEGF), stromal cell derived factor (SDF), and hepatocyte growth factor (HGF). In addition, spheroid culture promoted the preservation of extracellular matrix (ECM) components, such as laminin and fibronectin, in a culture time- and spheroid size-dependent manner. Furthermore, phosphorylation of AKT, a cell survival signal, was significantly higher and the expression of pro-apoptotic molecules, poly (ADP ribose) polymerase-1 (PARP-1) and cleaved caspase-3, was markedly lower in the spheroids than in MSCs in monolayers. In the murine hindlimb ischemia model, transplanted MSC spheroids showed better proliferation than MSCs in monolayer. These findings suggest that MSC spheroids promote MSC bioactivities via secretion of angiogenic cytokines, preservation of ECM components, and regulation of apoptotic signals. Therefore, MSC spheroid-based cell therapy may serve as a simple and effective strategy for regenerative medicine.

미세먼지는 다양한 건강 위해성을 유발하는 대표적인 환경 오염 물질로 알려져 있으나, 안구 표면에 미치는 영향과 그 분자적 기전에 대해서는 아직 충분히 규명되지 않았다. 본 연구에서는 각막 상피세포 유래 3차원 spheroid 모델에서 PM2.5가 spheroid의 안정성에 미치는 영향을 조사하였다. 각막 상피세포 유래 spheroid에 PM2.5를 처리한 결과, 세포 생존율의 유의한 변화 없이 spheroid의 형태학적 변화가 관찰되어 PM2.5가 비치명적 세포 스트레스를 유도함을 확인하였다. 이러한 조건에서도 PM2.5는 각막 상피 장벽 기능을 현저히 저해하였으며, 이는 TEER의 감소 및 ZO-1, ZO-3, occludin과 같은 밀착 연접 단백질 발현 감소로 입증되었다. 또한 PM2.5 노출은 MMP-1, -2 및 -3의 발현과 MMP-2 분비를 증가시켜 기질 분해 활성화를 유도하였다. 기전적으로, PM2.5는 미토콘드리아 및 소포체 스트레스를 유발하고 세포 내 칼슘 항상성을 교란하였으며, ER 스트레스 및 자가포식 관련 지표의 변화를 동반하였다. 더 나아가 PM2.5는 염증성 사이토카인의 발현을 증가시키고, 미토콘드리아에서 세포질로의 cytochrome c 이동과 MAPK 및 Akt 신호 경로 변화를 유도하였다. 종합적으로, 본 연구는 PM2.5가 세포 독성을 동반하지 않는 조건에서도 각막 상피의 구조적 재편성, 장벽 기능 손상, 세포 소기관 스트레스 및 염증 반응을 연속적으로 유도함을 제시하며, 환경 미세먼지 노출이 안구 표면 기능 이상에 기여할 수 있는 분자적 기전을 제공한다.

Particulate matter (PM) is a major environmental pollutant associated with diverse adverse health effects; however, its impact on the ocular surface and its underlying mechanisms are incompletely understood. In this study, we investigated the effects of diesel-derived PM2.5 (diameter ≤2.5 ㎛) on human corneal epithelial cells using a three-dimensional spheroid model that more accurately mimics in vivo tissue structure. Exposure of corneal epithelial spheroids to PM2.5 (25-50 ㎍/mL) induced marked morphological changes without significantly affecting cell viability, indicating sublethal cellular stress. Although no apparent cytotoxicity was observed, PM2.5 markedly impaired epithelial barrier function, as evidenced by reduced transepithelial electrical resistance and decreased expression of tight junction-related proteins. These changes were accompanied by increased expression and secretion of matrix metalloproteinases, suggesting improved extracellular matrix remodeling. Mechanistically, PM2.5 exposure induced mitochondrial and endoplasmic reticulum (ER) stress, disrupted intracellular calcium homeostasis, and altered the expression of ER stress- and autophagy-related markers. Furthermore, PM2.5 significantly increased proinflammatory cytokine levels, promoted cytochrome c redistribution from mitochondria to the cytoplasm, and activated mitogen-activated protein kinases and Akt signaling pathways. Collectively, these results demonstrate that PM2.5 induces corneal epithelial dysfunction through complex structural, barrier, organelle, and inflammatory alterations under noncytotoxic conditions. This study provides mechanistic insights into how environmental PM2.5 exposure contributes to ocular surface dysfunction and illustrates the utility of a 3D corneal epithelial model for assessing sublethal environmental toxicity.

This paper shows added mass and inertia can be acquired from the pure heaving motion and pure pitching motion respectively. A Vertical Planar Motion Mechanism (VPMM) test for the spheroid-type Unmanned Underwater Vehicle (UUV) was compared with a theoretical calculation and Computational Fluid Dynamics (CFD) analysis in this paper. The VPMM test has been carried out at a towing tank with specially manufactured equipment. The linear equations of motion on the vertical plane were considered for theoretical calculation, and CFD results were obtained by commercial CFD package. The VPMM test results show good agreement with theoretical calculations and the CFD results, so that the applicability of the VPMM equipment for an underwater vehicle can be verified with a sufficient accuracy.

Variations in adriamycin uptake and cytotoxicity were studied in tumor cells that were grown in different growth states and microenvironments. RIF-1 tumor cells were maintained in an RPMI 1640 medium, and grown in either a monolayer or multicell spheroids. For exponentially growing cells, adriamycin cytotoxicity increased with increased dosage up to 2.5 $\mu$g/ml, and this cytotoxicity was reduced when the cells were grown in a plateau phase or in an acidic microenvironment (pH 6.6). This reduced cytotoxicity was correlated with the uptake of the drug. For multicell spheroids, the cytotoxicity of the drug was reduced dramatically, and this reduction was also correlated with a reduced uptake of the drug and an acidic pH inside of the spheroids. When the drug cytotoxicity was evaluated at different locations within the spheroids, the cells in the inner regions were least affected by the drug, suggesting that both an acidic microenvironment and noncycling plateau phase cells are contributing factors in decreasing the efficacy of the drug in an organized tissue, such as multicell spheroids.