Question 1

Bioprocessing effect of carbon dioxide: Excess CO2 can suppress cell growth and productivity primarily by which mechanism(s)?

Accepted Answer

Introduction / Context: In mammalian and microbial cell culture, dissolved CO2 is a critical process parameter. Elevated CO2 partial pressure impacts cellular physiology and product quality, making its control essential in bioreactors and large-scale bioprocesses. Given Data / Assumptions: CO2 is membrane-permeable and hydrates to carbonic acid inside cells. Intracellular pH and carbamylation reactions can modulate enzyme function and metabolism. High CO2 may also depress respiration and energy metabolism. Concept / Approach: CO2 enters cells, forming carbonic acid and bicarbonate, lowering intracellular pH and altering acid–base balance. Carbamate formation on proteins can change activity. In parallel, respiratory enzymes and mitochondrial function can be downregulated by hypercapnic conditions. While medium pH shifts occur, well-buffered media can mask external pH changes even when intracellular perturbation remains significant. Step-by-Step Solution: Recognize two major intracellular effects: pH acidification and metabolic/respiratory inhibition.Acknowledge that external medium pH is not the only determinant; intracellular CO2 chemistry is pivotal.Choose the option reflecting both mechanisms. Verification / Alternative check: Process literature shows correlations between high pCO2 and reduced growth/productivity, reversible by CO2 stripping or base addition with careful control of intracellular pH indicators. Why Other Options Are Wrong: Only respiration or only medium pH does not capture the full effect. CO2 does not drive dissolved oxygen to zero; O2 solubility is governed mainly by its own partial pressure and temperature. Common Pitfalls: Assuming controlling bulk pH alone eliminates CO2 stress; overlooking intracellular acidification despite extracellular pH control. Final Answer: Both inhibiting respiration and altering intracellular pH

Question 2

Cell culture formats: Which statement correctly compares primary cell lines (PCL) and established cell lines (ECL) with respect to suspension culture?

Accepted Answer

Introduction / Context: Adherent versus suspension growth is a key practical distinction in cell culture. Many industrial processes prefer suspension-capable lines for scalability in bioreactors. This question distinguishes the adaptability of primary versus established lines to suspension culture. Given Data / Assumptions: Primary cell lines (PCL) are freshly isolated, finite, and often anchorage-dependent. Established cell lines (ECL) are immortalized or transformed and frequently adapt to suspension or reduced anchorage dependence. Concept / Approach: Most PCLs require attachment (e.g., via extracellular matrix or surface proteins) and exhibit contact inhibition and limited lifespan, making suspension culture unusual. Conversely, many ECLs (e.g., HEK293 derivatives, CHO, hybridomas) can be selected or adapted for suspension growth, enabling large-scale production in stirred-tank bioreactors. Step-by-Step Solution: Contrast anchorage dependence of PCL with adaptability of ECL.Relate industrial practice: ECLs commonly run in suspension for protein, vaccine, or viral vector production.Select the statement reflecting this reality. Verification / Alternative check: Manufacturing platforms (CHO-S, 293-F) demonstrate stable suspension growth and high-density culture, while PCLs (e.g., primary fibroblasts) typically demand adherent culture with serum and matrices. Why Other Options Are Wrong: Claiming both or only primary lines are commonly suspension-capable is inaccurate. Asserting neither or only hybridomas grow in suspension ignores numerous ECL examples. Common Pitfalls: Assuming any mammalian cell can be trivially adapted to suspension; overlooking the specialized selection needed for some ECLs. Final Answer: Established cell lines can be maintained in suspension cultures, whereas this is exceptional for primary cell lines

Question 3

Tissue dissociation challenge: During isolation of free cells and cell aggregates from solid organs, what is the major technical problem to overcome?

Accepted Answer

Introduction / Context: Preparing primary cell suspensions from tissues (liver, kidney, tumors) requires breaking cell–cell junctions and cell–matrix adhesions while preserving cell viability and functionality. This is foundational for primary culture, single-cell analyses, and regenerative studies. Given Data / Assumptions: Solid organs contain extensive extracellular matrix (ECM) and tight junctional complexes. Target outcome is viable single cells or small aggregates. Concept / Approach: The major challenge is to release cells from the ECM and from each other without excessive shear or proteolytic damage. Enzymes such as collagenase, dispase, elastase, and trypsin, plus chelators (EDTA/EGTA) and mechanical trituration, are used carefully to disrupt ECM and adhesions. Over-digestion or harsh shear compromises viability and surface receptors. Step-by-Step Solution: Define the goal: obtain free cells/aggregates while preserving viability.Identify the barrier: strong ECM and adhesion molecules (collagen, laminin, integrins, cadherins).Apply controlled enzymatic and mechanical methods to release cells from the matrix. Verification / Alternative check: Quality checks include trypan blue viability, yield counts, and functional assays; optimizing enzyme concentration, temperature, and time improves outcomes. Why Other Options Are Wrong: “Inhibiting” detachment contradicts the goal. “Disintegrating cells” destroys the preparation. Avoiding enzymes/chelators entirely is unrealistic for most tissues. Removing organelles is neither feasible nor relevant. Common Pitfalls: Overexposure to proteases, insufficient washing, and excessive shear leading to low viability or altered phenotype; ignoring tissue-specific protocols. Final Answer: Releasing the cells from their supporting extracellular matrix without damaging viability

Question 4

Animal cell culture — identify the main constituents required in the medium to support robust cell growth and division.  Consider carbon/energy sources (e.g., glucose and glutamine) as well as signaling supplements (growth factors and cytokines). Ch…

Accepted Answer

Introduction / Context: Animal cells grown in vitro require carefully formulated media that provide energy, biosynthetic precursors, and extracellular signals. Understanding what a “complete” medium contains is fundamental in cell culture and bioprocess engineering. Given Data / Assumptions: Candidates include metabolic substrates (glucose, glutamine) and regulatory additives (growth factors and cytokines). We assume standard mammalian culture for proliferation rather than minimal survival only. Balanced salts and pH buffers are necessary but not sufficient for sustained growth. Concept / Approach: Cells need both nutrients and signals. Glucose provides ATP via glycolysis and oxidative pathways. Glutamine fuels the TCA cycle and nitrogen metabolism. Growth factors (such as insulin, EGF, FGF) and selected cytokines convey mitogenic and survival signals through receptor pathways (MAPK/ERK, PI3K/AKT), enabling progression through the cell cycle and preventing apoptosis. Therefore, a complete answer must encompass all categories. Step-by-Step Solution: Identify core carbon/nitrogen sources: glucose + glutamine.Identify signal molecules: growth factors and, where relevant, cytokines.Recognize that salts/buffers alone cannot support proliferation.Select the option that includes nutrients and signals: “All of these.” Verification / Alternative check: Common media (DMEM, RPMI-1640) are typically supplemented with serum (a source of many growth factors and cytokines) or with defined recombinant factors in serum-free formulations, plus glucose and glutamine or stabilized dipeptides. Why Other Options Are Wrong: Glucose and glutamine alone lack mitogenic signals; growth factors or cytokines alone cannot provide metabolic substrates; salts/buffers maintain osmolality and pH but not growth. Common Pitfalls: Assuming serum alone is sufficient without attention to glucose/glutamine stability; overlooking that glutamine can degrade, requiring monitoring or use of stable dipeptides (e.g., alanyl-glutamine). Final Answer: All of these

Question 5

Cell line longevity — what is the term for cell lines that acquire the ability to be subcultured indefinitely in vitro (beyond the finite lifespan of primary cells)?

Accepted Answer

Introduction / Context: Most primary mammalian cells have a limited replicative lifespan (the Hayflick limit). Some lines bypass this limit and can be maintained indefinitely. Correct terminology is essential for experimental design, quality control, and regulatory documentation. Given Data / Assumptions: The question describes cells that can be subcultured indefinitely. No specific species or transformation mechanism is provided. We distinguish among primary, secondary, and continuous (established) lines. Concept / Approach: Cells that proliferate without senescence under culture conditions are typically called “established” or “continuous” cell lines. They may arise via spontaneous immortalization, viral oncogenes, or engineered telomerase expression. Primary and secondary lines are finite and senesce after limited passages. Step-by-Step Solution: Match the definition “indefinite subculture” to accepted nomenclature.Recall examples: HeLa, HEK293 (transformed/immortalized) are established lines.Exclude finite categories (primary/secondary).Select “Established cell lines (continuous cell lines).” Verification / Alternative check: Cell culture texts and ATCC documentation consistently use “continuous/established” for immortal cell populations capable of unlimited passages. Why Other Options Are Wrong: Primary and secondary are finite; “propagated cell lines” is nonspecific; terminally differentiated cultures do not divide indefinitely. Common Pitfalls: Confusing “long-term culture” with true immortalization; assuming serum concentration alone determines lifespan. Final Answer: Established cell lines (continuous cell lines)

Question 6

Media formulation — what is the typical optimum range of L-glutamine concentration used in mammalian cell culture media for growth and productivity?

Accepted Answer

Introduction / Context: L-glutamine is a pivotal amino acid in mammalian culture, serving as both a nitrogen donor and an anaplerotic carbon source. Its concentration strongly influences growth rate, lactate/ammonia by-products, and recombinant protein yield. Given Data / Assumptions: Question asks for an optimum range, not a single exact value. Common basal media provide 2–4 mmol/L glutamine; higher levels may be used with stabilized dipeptides. Glutamine is unstable in solution, degrading to ammonia over time. Concept / Approach: Practical “sweet spots” for many CHO and hybridoma processes sit around 2–6 mmol/L, balancing sufficient anaplerosis with manageable ammonia load. Thus, 2–7 mmol/L best reflects the broadly accepted, workable window across diverse cell types and processes. Step-by-Step Solution: Review standard media (DMEM, RPMI, MEM) and serum-free feeds.Note typical supplementation at ~2–4 mmol/L; fed-batch may increase within mid-single-digit mmol/L.Exclude very low (suboptimal) and very high (toxic/ammoniagenic) ranges.Select 2–7 mmol/L as the optimal range. Verification / Alternative check: Process development routinely titrates glutamine within 2–6 mmol/L, or uses stable dipeptides (e.g., 2–8 mmol/L equivalent) to reduce ammonia formation while maintaining flux. Why Other Options Are Wrong: 1–2 mmol/L can be limiting; 7–15 or 15–20 mmol/L risks excessive ammonia and acid load; 0.1–0.5 mmol/L is insufficient for most proliferative cultures. Common Pitfalls: Ignoring glutamine decay; not adjusting feed strategies in high-density cultures; conflating glutamine with glutamate (transport/usage differ). Final Answer: 2–7 mmol/L

Question 7

Bioenergetics of working muscle — which listed compound is NOT a direct source of usable energy for active muscle cells?

Accepted Answer

Introduction / Context: During intense exercise, muscle fibers rely on immediate and short-term energy systems: ATP, phosphocreatine, anaerobic glycolysis, and aerobic metabolism of glucose and fatty acids. Clarifying which molecules actually supply usable energy in the moment helps interpret fatigue and metabolite measurements. Given Data / Assumptions: Active muscle requires rapid ATP turnover. Phosphocreatine buffers ATP via creatine kinase. Glucose and fatty acids fuel ATP production via glycolysis and oxidative phosphorylation. Lactate accumulates during high glycolytic flux but is not oxidized significantly by the same fiber during peak anaerobic output. Concept / Approach: ATP is the immediate energy currency. Creatine phosphate rapidly regenerates ATP. Glucose and fatty acids are substrates for ATP generation. Lactate is primarily an end-product of anaerobic glycolysis under oxygen-limited conditions and is exported; it can be metabolized later (e.g., in liver via the Cori cycle or in oxidative fibers/heart) but is not the primary contemporaneous energy donor at the moment of its formation in active anaerobic fibers. Step-by-Step Solution: Identify immediate ATP sources: ATP and phosphocreatine.Identify metabolic fuels: glucose and fatty acids.Recognize lactate as a by-product/temporary sink for reducing equivalents.Therefore lactate is not a direct energy source in active contracting fibers. Verification / Alternative check: Physiology texts describe lactate shuttling and subsequent utilization by other tissues or later time points, not as the principal contemporaneous fuel within glycolytic bursts. Why Other Options Are Wrong: ATP powers myosin ATPase; phosphocreatine buffers ATP; glucose and fatty acids provide ATP via metabolic pathways. Common Pitfalls: Equating “can be metabolized somewhere later” with “is a present energy source.” Final Answer: Lactic acid (lactate)

Question 8

Human fibroblast cultures — these are best classified as which type of cell line with finite lifespan and stable characteristics?

Accepted Answer

Introduction / Context: Human dermal fibroblasts are a classic model for primary, finite cell culture. They divide for a limited number of passages before senescence. Distinguishing finite primary cultures from established (immortal) lines is important for experimental design and interpretation. Given Data / Assumptions: Example given: human fibroblast. Typical behavior: finite doubling potential (Hayflick limit) and contact inhibition. No evidence of transformation or immortalization stated. Concept / Approach: Primary fibroblasts are finite, genetically stable compared to transformed lines, and maintain many physiological characteristics. They are not “established” (immortal) unless specifically transformed or telomerase-extended. They are not malignant and are distinct from pluripotent stem cell lines. Step-by-Step Solution: Identify fibroblast as a prototypical primary culture.Note finite passage number and senescence.Exclude categories implying immortality or transformation.Choose “Stable primary cell lines (finite lifespan).” Verification / Alternative check: Standard cell culture references classify human fibroblasts as finite primary or “normal diploid” cell lines with predictable senescence. Why Other Options Are Wrong: Established lines are immortal; “cell transformations” implies oncogenic change; iPSC lines are reprogrammed pluripotent and behave differently. Common Pitfalls: Assuming “stable” means immortal; in cell culture, “finite lifespan” and genetic stability characterize primary fibroblasts. Final Answer: Stable primary cell lines (finite lifespan)

Question 9

Effect of oxygen limitation — when dissolved oxygen (DO) drops below the critical level in mammalian culture, which metabolic changes drive the decline in viable cell density?

Accepted Answer

Introduction / Context: Oxygen transfer is a key constraint in high-density animal cell culture. When DO falls below a critical value, cells rewire metabolism, often decreasing productivity and viability. Understanding the linked glutamine and glucose pathways helps troubleshoot bioreactor performance. Given Data / Assumptions: Critical DO is the threshold below which oxygen becomes limiting for aerobic metabolism. Glutamine and glucose are primary fuels; their catabolism affects by-products (ammonia, lactate). Observed outcome: decline in viable cell concentration. Concept / Approach: With insufficient oxygen, cells reduce oxidative metabolism, increasing glycolytic flux and lactate formation from glucose. In parallel, glutamine is incompletely oxidized, contributing to by-product accumulation and pH/ammonia stress. Together, these metabolic shifts burden cells, decrease growth, and promote apoptosis/necrosis. Step-by-Step Solution: Recognize oxygen limitation → shift to anaerobic glycolysis → lactate rise.Note glutaminolysis becomes inefficient → ammonia and acid load increase.Both processes stress cells and reduce viability.Select “Both (a) and (b).” Verification / Alternative check: Bioprocess data typically show negative correlations between prolonged low DO and viability, accompanied by elevated lactate and ammonia, especially at high cell densities. Why Other Options Are Wrong: Ammonia accumulation is largely coupled to glutamine metabolism (so “only, unrelated” is incorrect); ROS usually decrease when O2 is low, not rise from “excessive oxidative phosphorylation.” Common Pitfalls: Blaming a single metabolite only; ignoring that oxygen limitation simultaneously impacts multiple pathways. Final Answer: Both (a) and (b)

Question 10

Diagnosing unhealthy animal cell cultures — if large amounts of lactic acid accumulate in the culture fluid, what is the most likely underlying problem?

Accepted Answer

Introduction / Context: Elevated lactate in mammalian culture indicates reliance on glycolysis with pyruvate reduction to lactate instead of mitochondrial oxidation. This often signals oxygen limitation or excessive glycolytic feeding, both of which can harm culture performance. Given Data / Assumptions: Observation: substantial lactic acid accumulation in spent media. We are dealing with animal cells, not yeast (so ethanol is not expected). We assume typical glucose-containing media. Concept / Approach: Under low DO, pyruvate from glycolysis is reduced to lactate to regenerate NAD+, sustaining ATP production via glycolysis. This shift leads to acidification, reduced productivity, and potential apoptosis if prolonged. Therefore oxygen limitation is the most probable root cause. Step-by-Step Solution: Connect high lactate → anaerobic glycolysis.Identify the trigger: insufficient oxygen availability.Exclude alternatives inconsistent with mammalian metabolism (ethanol production).Select “Cells do not have enough oxygen.” Verification / Alternative check: Monitoring DO, OUR/OUR to KLa balance, and pH profiles typically confirms oxygen limitation coinciding with lactate spikes. Why Other Options Are Wrong: Mammalian cells do not produce ethanol; hyperoxia would favor oxidation and lower lactate; glycolysis inhibition would decrease, not increase, lactate; glutamine does not force “only oxidative” metabolism. Common Pitfalls: Attributing lactate solely to overfeeding rather than also considering oxygen transfer limits; ignoring mixing and KLa constraints in larger vessels. Final Answer: Cells do not have enough oxygen, driving anaerobic glycolysis

Question 11

Phenotypic shift after transformation — transformed mammalian cells frequently acquire which growth property regarding substrate attachment?

Accepted Answer

Introduction / Context: Cell transformation—whether spontaneous, viral, or oncogene-driven—often alters cytoskeletal regulation, adhesion, and signaling, producing hallmark traits used to assess tumorigenicity in vitro. Given Data / Assumptions: We focus on growth behavior with respect to attachment. Transformed cells commonly lose strict requirements for anchorage and contact inhibition. Soft agar colony formation is a classic assay of transformation. Concept / Approach: Normal epithelial and many mesenchymal cells are anchorage dependent. Transformation frequently confers anchorage independence, allowing proliferation in semisolid media (soft agar) and enabling growth in suspension—a phenotype associated with malignancy and metastatic potential. Step-by-Step Solution: Recall transformation hallmarks: loss of contact inhibition, anchorage independence.Map to assays: soft agar colony formation tests anchorage independence.Select the option that matches these phenotypes.Answer: anchorage independent growth. Verification / Alternative check: Oncogenic Ras/Myc and SV40 large T antigen transformations routinely convert cells to anchorage-independent phenotypes. Why Other Options Are Wrong: Anchorage dependency characterizes non-transformed cells; “enhanced genomic stability” is the opposite of typical transformation; “pronounced instability preventing any colony formation” is not a standard hallmark; feeder layers are not specific to transformation status. Common Pitfalls: Equating fast growth with transformation without testing anchorage independence. Final Answer: Anchorage independent growth (e.g., growth in soft agar)

Question 12

Cytogenetic diagnosis — which conditions can be detected by karyotyping as an unbalanced chromosomal complement?

Accepted Answer

Introduction / Context: Karyotyping visualizes whole chromosomes to detect numerical abnormalities and large structural rearrangements. It remains a cornerstone for diagnosing aneuploidies and sex chromosome disorders. Given Data / Assumptions: We are asked about conditions resulting from unbalanced complements. Examples include autosomal trisomy and sex chromosome aneuploidies. Karyotyping’s resolution is on the order of several megabases, not tiny microdeletions. Concept / Approach: Down’s (47, +21), Turner’s (45, X), and Klinefelter’s (47, XXY) are classic diagnoses by karyotype, clearly showing gains or losses of whole chromosomes or sex chromosomal complements. Step-by-Step Solution: List candidate disorders and their cytogenetic signatures.Confirm that each is a whole-chromosome aneuploidy.Recognize detectability via standard metaphase karyotyping.Select “All of these.” Verification / Alternative check: Clinical cytogenetics routinely confirms these diagnoses with karyogram analysis; additional techniques (FISH, microarrays) complement but are not required for whole-chromosome changes. Why Other Options Are Wrong: “Only microdeletions below 100 kb” are generally below karyotype resolution and require higher-resolution methods. Common Pitfalls: Confusing karyotyping’s strengths (aneuploidy) with its limitations (small CNVs). Final Answer: All of these

Question 13

Plasma choice for clotting in culture — why is adult chicken plasma preferred over mammalian plasma in some tissue culture setups?

Accepted Answer

Introduction / Context: Historically, plasma clots were used as a substrate for explant and primary cell culture. The clarity and firmness of the clot influence observation, nutrient diffusion, and handling. Given Data / Assumptions: Adult chicken plasma is compared with mammalian plasma. Desired features: clarity and firm clot formation even when diluted by medium. Use case: supporting adherent explants while allowing microscopy. Concept / Approach: Adult chicken plasma reliably forms a clear, firm coagulum when mixed with culture medium and coagulated (e.g., with thrombin or via clotting cascade). This property makes it advantageous for preparing stable plasma clots as supportive matrices that remain transparent for microscopic examination. Step-by-Step Solution: Identify the performance criteria: firmness and optical clarity.Relate to chicken plasma behavior: solid, transparent clot upon dilution.Exclude options inconsistent with historical practice.Select the correct rationale: clear, solid coagulum after dilution. Verification / Alternative check: Classical tissue culture protocols (pre-synthetic matrices) cite chicken plasma for consistent clear clotting, facilitating cell attachment and observation. Why Other Options Are Wrong: Opacity is not desirable; “no clots” or “semi-solid only” contradict use as a structural matrix; trypsin inactivation is not the key differentiator in this context. Common Pitfalls: Assuming all plasmas behave the same upon dilution with medium; not considering microscopy needs. Final Answer: It forms a clear, solid coagulum even after dilution with medium

Question 14

In animal cell culture (especially mammalian systems), what does the term “transformation” specifically mean? Provide the best interpretation used in cell biology laboratories rather than in bacterial genetics.

Accepted Answer

Introduction / Context: In mammalian cell culture, the vocabulary differs from microbial genetics. The word “transformation” is often confused with DNA uptake. In bacteria, transformation means acquisition of exogenous DNA. In mammalian cell biology, however, transformation generally refers to phenotypic changes that confer indefinite proliferation and tumor-like characteristics (loss of contact inhibition, altered morphology, anchorage-independent growth). Given Data / Assumptions: The setting is animal (mammalian) cell culture terminology. We must choose the cell biology meaning, not the bacterial genetics meaning. Typical transformed traits include faster doubling, altered karyotype, and reduced growth controls. Concept / Approach: Disambiguate terms: in mammalian culture, DNA delivery is properly called transfection (chemical, lipid, or electroporation methods). “Transformation” refers to the acquisition of neoplastic characteristics, either spontaneously or by oncogene/virus exposure. Therefore, the correct choice is the phenotypic change definition. Step-by-Step Solution: Identify domain: mammalian cell culture.Map terms: transformation = neoplastic-like change; transfection = DNA uptake.Select the option aligned with mammalian terminology. Verification / Alternative check: Standard texts describe “transformed” lines as continuous/immortalized, often aneuploid, with reduced contact inhibition and altered morphology. Why Other Options Are Wrong: A: DNA uptake in mammalian cells is called transfection, not transformation. C: Mixing definitions causes confusion; only (b) fits mammalian usage. D/E: Not relevant to accepted terminology. Common Pitfalls: Applying bacterial terms to mammalian contexts; assuming any genetic change equals transformation. Final Answer: Phenotypic modification of cultured cells toward a neoplastic or indefinite-growth state

Question 15

In mammalian cell culture metabolism, which pair correctly lists the principal metabolic products derived from glucose and glutamine, respectively?

Accepted Answer

Introduction / Context: Industrial and research mammalian cell cultures (e.g., CHO, HEK293, hybridomas) exhibit characteristic carbon–nitrogen metabolism. Understanding the major by-products is critical for medium design and bioreactor control because these metabolites affect pH, osmolality, and cell viability. Given Data / Assumptions: Glucose is frequently metabolized via aerobic glycolysis (Warburg-like effect). Glutamine is a key nitrogen and carbon source; it deaminates to produce ammonium. We seek the dominant measurable products in typical cultures. Concept / Approach: Glucose in rapidly proliferating mammalian cells often converts to lactate even in the presence of oxygen (overflow metabolism), while glutamine deamination produces ammonium (NH4+), which accumulates and can be toxic at elevated levels. Although CO2 is produced in respiration, the signature pair emphasized in bioprocess control is lactate (from glucose) and ammonium (from glutamine). Step-by-Step Solution: Associate glucose metabolism → lactate accumulation in culture supernatant.Associate glutamine catabolism → ammonium release via glutaminase pathways.Select the option pairing lactate with ammonium. Verification / Alternative check: Bioreactor profiles routinely monitor lactate and ammonium; feeding strategies often target lowering their accumulation (e.g., controlled glutamine feeding). Why Other Options Are Wrong: A: CO2 and NH3 are produced but the primary routine pair is lactate and ammonium; NH3 exists largely as NH4+ at culture pH. B/D: Missing ammonium from glutamine; lactate alone is incomplete. E: Acetate/nitrate are not the hallmark products for these substrates in mammalian cells. Common Pitfalls: Ignoring ammonium toxicity; assuming strictly oxidative metabolism for glucose under all conditions. Final Answer: Lactate and ammonium

Question 16

Cell line status criteria: an “established” (continuous) mammalian cell line is traditionally recognized after extensive subculture. Which benchmark (commonly cited in classic lab practice) indicates establishment?

Accepted Answer

Introduction / Context: “Established” (continuous) cell lines are distinct from primary cultures and finite cell strains. Historically, labs used operational criteria—such as many consecutive subcultures without senescence—to denote that a culture has become continuous, typically accompanied by transformed characteristics. Given Data / Assumptions: Question refers to classic, widely taught benchmarks rather than a formal universal law. Established lines show indefinite propagation and often altered karyotypes. Intervals of ~3 days between passages are common in teaching examples. Concept / Approach: Although transformation/immortalization is the biological basis, many training manuals cite a conventional threshold (around 70 serial passages at typical intervals) to claim “establishment.” The option that matches this teaching convention is the one with 70 subcultures at ~3-day intervals. Step-by-Step Solution: Identify traditional benchmark used in many MCQ banks and lab manuals.Match passage number and interval to the conventional figure.Select 70 subcultures at ~3-day intervals. Verification / Alternative check: Many continuous lines (e.g., HeLa, certain CHO derivatives) propagate indefinitely and quickly surpass dozens of passages, fitting the “established” concept. Why Other Options Are Wrong: B/D: Lower passage counts are less stringent; used for strains rather than continuous status. C: Interval detail is atypical; not the commonly quoted pair. E: Far too few passages to infer establishment. Common Pitfalls: Assuming there is a strict universal passage-number definition; the essence is indefinite growth with transformed traits, but the 70-passage benchmark is a standard test answer. Final Answer: At least 70 subcultures at ~3-day intervals

Question 17

Animal tissue culture methods: which one of the following is NOT a classic “explantation” technique, but rather a general culture mode?

Accepted Answer

Introduction / Context: Explantation techniques are historical and classic methods for cultivating small pieces of tissue or organ fragments (explant cultures). They include formats like Carrel flasks, roller tubes, and similar setups designed specifically for maintaining tissue fragments. Given Data / Assumptions: “Explantation” emphasizes small tissue fragments maintained on or within a support (clots, gels, or surfaces). Carrel flask and roller tube systems are canonical explant/organ culture approaches. “Adherent primary culture” describes a general growth mode, not a specific explantation technique. Concept / Approach: We must distinguish named explant methods from broader descriptors. While slide culture can be used for certain tissue or microbial studies, and Carrel/roller formats are classic explant systems, “adherent primary culture” simply indicates cells growing attached to a surface—many culture types can be adherent and primary without being a named explant technique. Step-by-Step Solution: List the classic explantation techniques (e.g., Carrel flask, roller tube, hanging drop).Identify the option that is a generic description rather than a named method.Select “Adherent primary culture.” Verification / Alternative check: Standard references classify Carrel/roller/hanging-drop/plasma clot/raft as explant methods, whereas adherent/ suspension are general modes. Why Other Options Are Wrong: A/B/C/E: Each corresponds to a recognized explant culture setup or format. Common Pitfalls: Confusing “explant culture” (tissue pieces) with “primary culture” (cellular outgrowth), and using growth mode labels in place of technique names. Final Answer: Adherent primary culture

Question 18

Bioprocess design: what is an acceptable osmolality (mOsm/kg H2O) operating range commonly tolerated by mammalian cells in culture media?

Accepted Answer

Introduction / Context: Osmolality is a key media parameter affecting mammalian cell viability, productivity, and product quality (e.g., glycosylation). Cultures are typically maintained near physiologic osmolality, with some tolerance window depending on cell line and process strategy. Given Data / Assumptions: Physiological osmolality is roughly 280–300 mOsm/kg for many cells. Some fed-batch processes tolerate moderately higher osmolality (up to ~350–360 mOsm/kg). Extremes away from physiologic can impair growth and quality. Concept / Approach: Choose the range that captures the typical span seen in practical mammalian cell culture operations, allowing some headroom for feed additions and metabolite accumulation without causing severe stress. Step-by-Step Solution: Identify physiological target: ~280–300 mOsm/kg.Allow realistic process tolerance up to ~350–360 mOsm/kg.Select the option 280–360 mOsm/kg. Verification / Alternative check: Media recipes (e.g., DMEM variants) and process guidelines align with this range; product-quality studies often manage osmolality within this window. Why Other Options Are Wrong: A/E: Too low for typical mammalian systems; risks swelling and lysis. C: Narrow band that may be overly restrictive for fed-batch dynamics. D: Skews too high; many cell lines suffer above ~360 mOsm/kg. Common Pitfalls: Confusing osmolarity with osmolality; ignoring cumulative feed effects that raise osmolality. Final Answer: 280–360 mOsm/kg

Question 19

Specific oxygen consumption in mammalian cell culture: what is the typical per-cell rate range (qO2) expressed as pmol O2 per cell per hour?

Accepted Answer

Introduction / Context: The specific oxygen consumption rate (qO2) is critical for sizing aeration, agitation, and oxygen transfer in bioreactors. Mammalian cells consume far less oxygen per cell than microbes, so correct units and magnitudes matter in process design. Given Data / Assumptions: Mammalian qO2 values are very small on a per-cell basis. Reasonable design ranges are in picomoles O2 per cell per hour. nmol-per-cell values would be unrealistically high. Concept / Approach: Choose the range that fits literature-reported per-cell consumption. Values around 0.05–5 pmol O2/cell/h encompass many cell lines and physiological states, providing a practical order of magnitude for engineering calculations. Step-by-Step Solution: Check unit sanity: pmol-per-cell-per-hour is typical.Compare magnitudes: nmol-per-cell would imply orders-of-magnitude larger demand than observed.Select 0.05–5 pmol O2/cell/h as the realistic span. Verification / Alternative check: Back-of-the-envelope calculations for common viable cell densities (10^6–10^7 cells/mL) with this qO2 range align with measured oxygen transfer capacities in mammalian bioreactors. Why Other Options Are Wrong: A/B/E: nmol or mmol per cell per hour are far too high and physiologically implausible. D: The upper half overlaps but omits the lower portion commonly seen; the broader 0.05–5 pmol range is standard. Common Pitfalls: Mistaking per-cell rates for per-culture or per-mL rates; mixing up pmol and nmol units. Final Answer: 0.05–5 pmol O2/cell/h

Question 20

Culture media design: what is the typical optimum range for glucose concentration in mammalian cell culture media (expressed in mmol per litre)?

Accepted Answer

Introduction / Context: Glucose is a primary carbon source for mammalian cells. Standard media options range from “low glucose” (~5.5 mmol/L) to “high glucose” (~25 mmol/L). Understanding the practical concentration window informs feeding and control strategies to limit lactate accumulation. Given Data / Assumptions: Low-glucose DMEM is ~5.5 mmol/L; high-glucose DMEM is ~25 mmol/L. Custom and fed-batch processes may transiently reach higher values, but typical setpoints fall within a manageable range. Excess glucose can drive lactate overflow and acidification. Concept / Approach: Select the option that brackets common practice from low to reasonably elevated concentrations used in process development and routine culture. 5.5–55 mmol/L covers standard media and certain fed-batch excursions without implying extreme, generally undesirable levels. Step-by-Step Solution: Identify common baseline media concentrations (5.5 and ~25 mmol/L).Allow operational upper bounds under feed strategies.Pick the range 5.5–55 mmol/L. Verification / Alternative check: Manufacturer datasheets and process guidelines align with this operational window. Why Other Options Are Wrong: B–D: Excessively high, typically detrimental to culture health. E: Too low for standard mammalian cell growth. Common Pitfalls: Equating higher glucose with better growth; ignoring the lactate penalty and osmolality impacts. Final Answer: 5.5–55 mmol/L

Animal Cell Culture and Regulation Questions