Three-Dimensional Reconstructed Bone Marrow Matrix Culture Improves the Viability of Primary Myeloma Cells In-Vitro via a STAT3-Dependent Mechanism

Primary myeloma (PM) cells are short-lived in conventional culture, which limited their usefulness as a study model. Here, we evaluated if three-dimensional (3D) culture can significantly prolong the longevity of PM cells in-vitro. We employed a previously established 3D model for culture of bone marrow mononuclear cells isolated from 15 patients. We assessed the proportion of PM cells, viability and proliferation using CD38 staining, trypan blue exclusion assays and carboxy fluorescein succinimidyl ester (CFSE) staining, respectively. We observed significantly more CD38+ viable cells in 3D than in conventional culture (65% vs. 25%, p = 0.006) on day 3. CFSE staining showed no significant difference in cell proliferation between the two culture systems. Moreover, we found that PM cells in 3D culture are more STAT3 active by measure of pSTAT3 staining (66% vs. 10%, p = 0.008). Treatment of IL6, a STAT3 activator significantly increased CD38+ cell viability (41% to 68%, p = 0.021). In comparison, inhibition of STAT3 with Stattic significantly decreased PM cell viability in 3D culture (38% to 17% p = 0.010). Neither IL6 nor Stattic affected the PM cell viability in conventional culture. This study suggests that 3D culture can significantly improve the longevity of PM cells in-vitro, and STAT3 activation can further improve their viability.

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Effect of Campath-1H antibody on human hematopoietic progenitors in vitro

Blood ◽

10.1182/blood.v82.3.807.bloodjournal823807 ◽

1993 ◽

Vol 82 (3) ◽

pp. 807-812

Author(s):

MH Gilleece ◽

TM Dexter

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Fluorescein Isothiocyanate ◽

Lymphoid Cells ◽

Human Complement ◽

Hematopoietic Progenitors ◽

Pilot Studies ◽

Significant Difference ◽

And Control

The humanized antibody CAMPATH-1H has been shown in pilot studies to be beneficial in the treatment of lymphoid malignancy and other lymphoproliferative diseases. The antigen recognized by this antibody is not confined to lymphoid cells, and work with rat antibodies of similar specificity has not eliminated the possibility of damage to human hematopoietic progenitors, particularly those capable of repopulating bone marrow and sustaining hematopoiesis. This study aimed to discover if hematopoietic progenitor cells were affected by treatment with CAMPATH-1H, with or without human complement. Bone marrow mononuclear cells from healthy volunteers were treated with saturating concentrations of CAMPATH-1H, human complement, or CAMPATH- 1H plus human complement. The CD34-positive fraction of the mononuclear cells was treated similarly. Residual progenitor activity was measured in the colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte assay and compared with untreated controls. There was no significant difference (at the 5% level) between treated and control cells. Mononuclear cells were divided into CAMPATH-1H-positive and CAMPATH-1H-negative fractions by fluorescein isothiocyanate-CAMPATH-1H labeling and fluorescence-activated cell sorter separation. Hematopoietic progenitors were predominantly found in the CAMPATH-1H- negative fraction. Furthermore, mononuclear cells treated with CAMPATH- 1H and complement were equivalent to controls in experiments that investigated the capacity of these cells to form hematopoietic foci in long-term cultures.

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Effect of Campath-1H antibody on human hematopoietic progenitors in vitro

Blood ◽

10.1182/blood.v82.3.807.807 ◽

1993 ◽

Vol 82 (3) ◽

pp. 807-812 ◽

Cited By ~ 160

Author(s):

MH Gilleece ◽

TM Dexter

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Fluorescein Isothiocyanate ◽

Lymphoid Cells ◽

Human Complement ◽

Hematopoietic Progenitors ◽

Pilot Studies ◽

Significant Difference ◽

And Control

Abstract The humanized antibody CAMPATH-1H has been shown in pilot studies to be beneficial in the treatment of lymphoid malignancy and other lymphoproliferative diseases. The antigen recognized by this antibody is not confined to lymphoid cells, and work with rat antibodies of similar specificity has not eliminated the possibility of damage to human hematopoietic progenitors, particularly those capable of repopulating bone marrow and sustaining hematopoiesis. This study aimed to discover if hematopoietic progenitor cells were affected by treatment with CAMPATH-1H, with or without human complement. Bone marrow mononuclear cells from healthy volunteers were treated with saturating concentrations of CAMPATH-1H, human complement, or CAMPATH- 1H plus human complement. The CD34-positive fraction of the mononuclear cells was treated similarly. Residual progenitor activity was measured in the colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte assay and compared with untreated controls. There was no significant difference (at the 5% level) between treated and control cells. Mononuclear cells were divided into CAMPATH-1H-positive and CAMPATH-1H-negative fractions by fluorescein isothiocyanate-CAMPATH-1H labeling and fluorescence-activated cell sorter separation. Hematopoietic progenitors were predominantly found in the CAMPATH-1H- negative fraction. Furthermore, mononuclear cells treated with CAMPATH- 1H and complement were equivalent to controls in experiments that investigated the capacity of these cells to form hematopoietic foci in long-term cultures.

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Tumor-Activated Human NK Cells Specificall Ylyse Autologous and Allogeneic NK-Resistant Tumor Targets.

Blood ◽

10.1182/blood.v108.11.3707.3707 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3707-3707

Author(s):

Mark W. Lowdell ◽

Ismail Bakhsh ◽

Janet North ◽

Chloe Marden ◽

Elena Addison ◽

...

Keyword(s):

Breast Cancer ◽

Bone Marrow ◽

Nk Cells ◽

Tumor Cells ◽

Mononuclear Cells ◽

Leukemia Cell ◽

Lymphokine Activated Killer Cells ◽

Normal Peripheral Blood ◽

Significant Difference

Abstract Pre-incubation of resting human NK cells with the leukemia cell CTV-1 primes NK cells to lyse NK-resistant cell lines, primary leukemias and solid tumors, even when HLA-matched; allogeneic or autologous. The primed NK cells remained non-responsive to HLA-C matched and mismatched normal peripheral blood or bone marrow mononuclear cells from multiple donors and did not affect in vitro colony forming unit assays. NK cells isolated from normal healthy donors and co-incubated with irradiated CTV-1 tumor cells synthesized CD69 within 60 min with maximal expression achieved within 6 hr. The tumor-activated NK cells (T-ANKs) lysed NK resistant RAJI and Daudi cells in a 4-hour assay (Fig 1). The degree of lysis was equivalent to that of matched lymphokine activated killer cells (LAK) after non-specific activation with IL-2. CTV-1 cells are HLA-C type 2 homozygous and express HLA-Bw4 alleles. They thus can ligate KIR2DL1 (CD158a) and KIR3DL1 (CD158e1) on NK cells. Co-cultures of HLA-matched or mis-matched NK with CTV-1 showed there was no significant difference in the generation of T-ANK activity in the presence or absence of KIR ligation. T-ANK cells from allogeneic donors were capable of lysis of primary AML cells of all FAB types, the relatively NK-resistant breast cancer cell line, MCF-7, and primary tumor cells isolated from resected tissue from patients with breast cancer and ascities from patients with ovarian cancer. Autologous T-ANK cells were generated from five AML patients in clinical remission; all of whom had low level or undetectable NK activity to their presentation disease. T-ANK cells from all five donors consistently induced significantly greater lysis of autologous AML blasts than the matched NK cells (p<0.05) (Fig 2). To investigate the tumor-restriction of allogeneic T-ANK cells we isolated NK cells from normal donors and either activated them with CTV-1 cells overnight or maintained them in media. These T-ANK cells were then compared with NK cells from the same donor with respect to lysis of normal autologous and allogeneic PBMC. Neither NK nor T-ANK cells lysed autologous PBMC nor did they lyse PBMC from HLA-C mismatched normal donors. To determine the likelihood of bone marrow suppression by T-ANK cells we established hematopoietic colony forming assays with bone marrow from 5 normal donors and added T-ANK from HLA-C mismatched donors at increasing ratios. CFU-GM, BFU-E and CFU-GEMM were not affected by co-incubation with HLA-mismatched T-ANK even at effector:target ratios of 20:1. We believe that this demonstrates a two stage process for NK activation; priming and triggering, the ligands for which are distinct. Tumor priming of NK primes cells capable of lysing a broad spectrum of NK-resistant tumors which may irrespective of KIR ligation which may represent an “off-the-shelf” immunotherapy product. Fig 1 Fig 1. Fig 2 Fig 2.

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In Vitro Periodontal Ligament Cell Viability in Different Storage Media

Brazilian Dental Journal ◽

10.1590/0103-6440201602294 ◽

2016 ◽

Vol 27 (4) ◽

pp. 408-411 ◽

Cited By ~ 4

Author(s):

Meenakshi Sharma

Keyword(s):

Cell Viability ◽

Periodontal Ligament ◽

Egg White ◽

Periodontal Ligament Cells ◽

Periodontal Ligament Cell ◽

Trypan Blue Exclusion ◽

Storage Media ◽

Significant Difference ◽

Rice Water

Abstract The aim of this study was to evaluate the viability of periodontal ligament cells of avulsed teeth in three different storage media. Forty-five mature premolars extracted for orthodontic therapeutic purposes were randomly and equally divided into three groups according to the storage medium: milk (control), rice water and egg white. After placing extracted teeth for 30 min in storage media, the scrapings of the periodontal ligament (PDL) were collected in Falcon tubes containing collagenase in 2.5 mL of phosphate buffer saline and were incubated for 30 min and centrifuged for 5 min at 800 rpm. Cell viability was analyzed by Trypan blue exclusion. Rice water had a significantly higher number of viable cells compared to egg white and milk. There was no statistically significant difference between egg white and milk. Rice water may be able to maintain PDL cell viability of avulsed teeth better than egg white or milk.

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Does Needle Design Affect the Regenerative Potential of Bone Marrow Aspirate? An In Vitro Study

Life ◽

10.3390/life11080748 ◽

2021 ◽

Vol 11 (8) ◽

pp. 748

Author(s):

Nadia Feddahi ◽

Monika Herten ◽

Tjark Tassemeier ◽

Heike Rekasi ◽

Alexander Hackel ◽

...

Keyword(s):

Bone Marrow ◽

Iliac Crest ◽

Mononuclear Cells ◽

Bone Marrow Aspirate ◽

In Vitro Study ◽

Contralateral Side ◽

Disc Disease ◽

Significant Difference ◽

Spinal Disc

While autologous bone is still the gold standard for treatment of bone defects, its availability is limited. Sufficient numbers of mesenchymal stroma cells (MSC) may be an alternative. Small volumes of bone marrow aspirate (BMA) were harvested with two different needle systems comparing the yield and regenerative potency of the MSCs. BMA (10 mL) was aspirated from the posterior iliac crest of 12 patients with degenerative spinal disc disease using both needle systems in each patient: the Jamshidi needle (JAM) and on the contralateral side the Marrow Cellution® Needle (AMC). Number of mononuclear cells (MNCs) and regeneration capacity (colony-forming unit/CFU) were determined. MSCs were characterized for surface markers and their differentiation into trilineages. There was no significant difference between the two harvesting needles regarding the quantity of MNCs in BMA: 5.2 ± 1.8 × 109 MNC/mL for AMC vs. 4.8 ± 2.5 × 109 MNC/mL for JAM, p = 0.182. The quantity of CFUs per ml BMA was similar for both groups: 3717 ± 5556 for AMC and 4305 ± 5507 for JAM (p = 0.695). The potency of MSCs expressed as colony-forming potential per 106 MNC resulted in 0.98 ± 1.51 for AMC and 1.00 ± 0.96 for JAM (p = 0.666). Regardless of the needle design, 10 mL bone marrow aspirate contains a sufficient number of about 40,000 MSCs that can be used to enhance bone healing.

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3D Culture Modelling: An Emerging Approach for Translational Cancer Research in Sarcomas

Current Medicinal Chemistry ◽

10.2174/0929867326666191212162102 ◽

2020 ◽

Vol 27 (29) ◽

pp. 4778-4788 ◽

Cited By ~ 1

Author(s):

Victoria Heredia-Soto ◽

Andrés Redondo ◽

José Juan Pozo Kreilinger ◽

Virginia Martínez-Marín ◽

Alberto Berjón ◽

...

Keyword(s):

High Throughput Screening ◽

Cancer Biology ◽

Soft Tissues ◽

Three Dimensional ◽

3D Culture ◽

Resistance Mechanisms ◽

In Vitro Models ◽

Tumour Spheroids ◽

Current Therapies

Sarcomas are tumours of mesenchymal origin, which can arise in bone or soft tissues. They are rare but frequently quite aggressive and with a poor outcome. New approaches are needed to characterise these tumours and their resistance mechanisms to current therapies, responsible for tumour recurrence and treatment failure. This review is focused on the potential of three-dimensional (3D) in vitro models, including multicellular tumour spheroids (MCTS) and organoids, and the latest data about their utility for the study on important properties for tumour development. The use of spheroids as a particularly valuable alternative for compound high throughput screening (HTS) in different areas of cancer biology is also discussed, which enables the identification of new therapeutic opportunities in commonly resistant tumours.

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Suitability of a Progenitor Cell-Enriching Device for In Vitro Applications

Coatings ◽

10.3390/coatings11020146 ◽

2021 ◽

Vol 11 (2) ◽

pp. 146

Author(s):

Antonio Celentano ◽

Tami Yap ◽

Giuseppe Pantaleo ◽

Rita Paolini ◽

Michael McCullough ◽

...

Keyword(s):

Cell Viability ◽

Time Course ◽

Ex Vivo ◽

Trypan Blue Exclusion ◽

Trypan Blue Exclusion Test ◽

Initial Reduction ◽

Metal Wear ◽

Class 1 ◽

Electron Energy Loss

Rigenera® is a novel class-1 medical device that produces micro-grafts enriched of progenitors cells without ex vivo manipulation of donor tissues. The manufacturer’s protocol has been supported for a wide variety of clinical uses in the field of regenerative medicine. This study aimed to evaluate its potential use for in vitro cell models. Human primary oral fibroblasts were cultured under standard conditions and processed through Rigenera® over a time course of up to 5 min. Cell viability was assessed using a Trypan Blue exclusion test. It is possible to process fibroblasts through Rigenera® although an initial reduction of cell viability was observed. Additionally, debris was evident in the cell suspension of the processed samples. Scanning electron microscopy (SEM) microanalysis of the debris and electron energy-loss spectroscopy confirmed the presence of metal wear possibly due to the processing conditions used in this study. Interestingly, pore sizes within Rigeneracons® grids were found to range between 250–400 μm. This is the first report assessing the suitability of Rigenera® and Rigeneracons® for in vitro applications. Whilst Rigenera® workflow was found to be amenable to laboratory uses, our results strongly suggest that further research and development is necessary to support the utilization of this technology for enrichment of micro-graft derived cells and cell sorting in vitro.

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Biological characterization of implant surfaces - in vitro study

Revista de Odontologia da UNESP ◽

10.1590/1807-2577.1087 ◽

2015 ◽

Vol 44 (4) ◽

pp. 195-199 ◽

Cited By ~ 1

Author(s):

Priscilla Barbosa Ferreira Soares ◽

Camilla Christian Gomes Moura ◽

Huberth Alexandre da Rocha Júnior ◽

Paula Dechichi ◽

Darceny Zanetta-Barbosa

Keyword(s):

Alkaline Phosphatase ◽

Cell Viability ◽

Total Protein ◽

Cell Attachment ◽

Control Group ◽

Mitochondrial Activity ◽

Serum Total Protein ◽

Trypan Blue Exclusion ◽

Experimental Surface

<title>Abstract</title><sec><title>Objective</title>Evaluate the biological performance of titanium alloys grade IV under different surface treatments: sandblasting and double etching (Experimental surface 1; Exp1, NEODENT); surface with wettability increase (Experimental surface 2; Exp2, NEODENT) on response of preliminary differentiation and cell maturation.</sec><sec><title>Material and method</title>Immortalized osteoblast cells were plated on Exp1 and Exp2 titanium discs. The polystyrene plate surface without disc was used as control group (C). Cell viability was assessed by measuring mitochondrial activity (MTT) at 4 and 24 h (n = 5), cell attachment was performed using trypan blue exclusion within 4 hours (n = 5), serum total protein and alkaline phosphatase normalization was performed at 4, 7 and 14 days (n = 5). Data were analyzed using one-way ANOVA and Tukey test.</sec><sec><title>Result</title>The values of cell viability were: 4h: C– 0.32±0.01A; Exp1– 0.34±0.08A; Exp2– 0.29±0.03A. 24h: C– 0.43±0.02A; Exp1– 0.39±0.01A; Exp2– 0.37±0.03A. The cell adhesion counting was: C– 85±10A; Exp1- 35±5B; Exp2– 20±2B. The amounts of serum total protein were 4d: C– 40±2B; Exp1– 120±10A; Exp2– 130±20A. 7d: C– 38±2B; Exp1– 75±4A; Exp2– 70±6A. 14 d: C– 100±3A; Exp1– 130±5A; Exp2– 137±9A. The values of alkaline phosphatase normalization were: 4d: C– 2.0±0.1C; Exp1– 5.1±0.8B; Exp2– 9.8±2.0A. 7d: C– 1.0±0.01C; Exp1– 5.3±0.5A; Exp2– 3.0±0.3B. 14 d: C– 4.1±0.3A; Exp1– 4.4±0.8A; Exp2– 2.2±0.2B. Different letters related to statistical differences.</sec><sec><title>Conclusion</title>The surfaces tested exhibit different behavior at dosage of alkaline phosphatase normalization showing that the Exp2 is more associated with induction of cell differentiation process and that Exp1 is more related to the mineralization process.</sec>

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Size-controlled human adipose-derived stem cell spheroids hybridized with single-segmented nanofibers and their effect on viability and stem cell differentiation

Biomaterials Research ◽

10.1186/s40824-021-00215-9 ◽

2021 ◽

Vol 25 (1) ◽

Author(s):

Jinkyu Lee ◽

Sangmin Lee ◽

Sung Min Kim ◽

Heungsoo Shin

Keyword(s):

Stem Cell ◽

Cell Function ◽

Average Length ◽

Three Dimensional ◽

Stem Cell Differentiation ◽

Cell Spheroids ◽

Significant Difference ◽

Vitro Analysis ◽

Physical And Chemical

Abstract Background Fabrication of three-dimensional stem cell spheroids have been studied to improve stem cell function, but the hypoxic core and limited penetration of nutrients and signaling cues to the interior of the spheroid were challenges. The incorporation of polymers such as silica and gelatin in spheroids resulted in relatively relaxed assembly of composite spheroids, and enhancing transport of nutrient and biological gas. However, because of the low surface area between cells and since the polymers were heterogeneously distributed throughout the spheroid, these polymers cannot increase the cell to extracellular matrix interactions needed to support differentiation. Methods We developed the stem cell spheroids that incorporate poly(ι-lactic acid) single-segmented fibers synthesized by electrospinning and physical and chemical fragmentation. The proper mixing ratio was 2000 cells/μg fibers (average length of the fibers was 50 μm - 100 μm). The SFs were coated with polydopamine to increase cell binding affinity and to synthesize various-sized spheroids. The function of spheroids was investigated by in vitro analysis depending on their sizes. For statistical analysis, Graphpad Prism 5 software (San Diego, CA, USA) was used to perform one-way analysis of variance ANOVA with Tukey’s honest significant difference test and a Student’s t-test (for two variables) (P < 0.05). Results Spheroids of different sizes were created by modulating the amount of cells and fibers (0.063 mm2–0.322 mm2). The fibers in the spheroid were homogenously distributed and increased cell viability, while cell-only spheroids showed a loss of DNA contents, internal degradation, and many apoptotic signals. Furthermore, we investigated stemness and various functions of various-sized fiber-incorporated spheroids. In conclusion, the spheroid with the largest size showed the greatest release of angiogenic factors (released VEGF: 0.111 ± 0.004 pg/ng DNA), while the smallest size showed greater effects of osteogenic differentiation (mineralized calcium: 18.099 ± 0.271 ng/ng DNA). Conclusion The spheroids incorporating polydopamine coated single-segmented fibers showed enhanced viability regardless of sizes and increased their functionality by regulating the size of spheroids which may be used for various tissue reconstruction and therapeutic applications.

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