Day 8 part 1

Applied Molecular Cellular Biology

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🌟 Fun & Educational Summary — Cellular Milk, Plant Drinks, Precision Fermentation & Mammary Physiology

(Detailed, complete, theoretical-only)

🥛 1. Why Make Milk Without Cows?

Climate motivation

Denmark’s climate law (2020) requires 70% reduction of greenhouse gases by 2030.
Agriculture is a major contributor.
Methane = 35% of Danish greenhouse gases; 75% of methane comes from cows.
Methane is far more potent than CO₂ → reducing cow numbers or improving production efficiency matters.

Alternatives to conventional milk

🌱 Plant-based drinks → Not legally “milk.”
🧬 Precision fermentation drinks → Recombinant proteins from microbes.
🧫 Cell-based milk → Using mammary cells to produce actual milk components.

Future vision: hybrid products mixing plant, microbial, and cell-based components.

🌱 2. Plant-Based Drinks — Nutritional & Cellular Effects

Nutritional profile

Generally low in protein, have different amino acid profiles, and variable micronutrients.
Highly processed foods → stabilizers, emulsifiers, sugar, colors.

Study: Effect on intestinal cell viability

Setup:

12 plant drinks + 2 cow’s milk samples.
Human intestinal cells.
All samples processed to obtain whey-like phase (remove fat + casein equivalent).
Measure cell viability (resazurin / Alamar Blue assay).

Findings

🐄 Cow’s milk → clearly increases intestinal cell growth (expected due to growth factors).
🌾 Spelt drink → the only plant drink with similar stimulatory effect.
🌱 Soy → decreased cell viability.
🌾 Oat → mostly neutral/negative, one had a hormetic (“U-shaped”) response.
🥥 Coconut, almond, rice → generally no positive effect.

Conclusions

Plant drinks ≠ nutritional substitute for milk.
LACK: growth factors, bioactive peptides, complex lipid structures, etc.
Danish authorities: cannot replace milk 1:1, especially for children.
Some may also pose issues for diabetes risk due to processing and added sugar.

🧬 3. Precision Fermentation — Recombinant Milk Proteins

Concept

Insert cow milk protein genes into yeast/bacteria/fungi.
Ferment → cells produce individual proteins (caseins, whey proteins).
Harvest proteins → blend into beverages.

Companies

Perfect Day (USA), Tomorrow Farms, Remilk (Israel), Better Milk, etc.

Limitations

Milk has > 2,000 natural components: proteins, lactose, lipids, minerals, growth factors, hormones, exosomes, bioactives.
Precision-fermented drinks usually contain 6–10 proteins → essentially protein shakes, not milk.
Lacks natural complexity and biological functions of milk.

🧫 4. Cell-Based Milk — Using Mammary Cells Directly

Goal: grow mammary epithelial cells in vitro and induce them to secrete real milk containing the natural spectrum of components.

Why promising?

Mammary epithelial cells contain the full genetic program needed to produce nearly all milk components.

🐄 5. Mammary Gland Physiology — What Must Be Replicated in Culture

🔸 A. Development from heifer → cow

Heifers reach puberty at 250–280 kg.
Insemination: 13–16 months.
Calving after 9 months → lactation begins.
Cows are re-inseminated 2 months post-calving to maintain one calf/year.
Dry period of ~2 months before next calving.

📌 Why this matters: The physiological stage affects what mammary cells can produce. Cell sourcing must match productive stages.

🔸 B. Mammary gland structure

Heifer:

Small gland.
30–40% fat.
Few epithelial cells.

Cow:

Large gland (25–30 kg at slaughter).
Extensive lobulo-alveolar structure.
High content of milk-producing parenchymal tissue.

📌 Important because: Researchers must obtain parenchyma, not fat or duct tissue.

🔸 C. Hormonal regulation — essential for in vitro protocols

Key hormones:

Estrogen → drives ductal and alveolar growth. Removal of ovaries → gland becomes mostly fat (classic ovaryectomy experiment).
Growth hormone → expands parenchymal mass; used in some countries to increase milk yield.
Progesterone, prolactin, IGF-1, and others play roles in alveolar development and lactogenesis.

Cells require precise exposure to these hormones to differentiate and secrete milk.

🔸 D. Milk synthesis pathways

Milk components derive from:

Glucose → lactose synthesis.
Amino acids → caseins, whey proteins.
Acetate / fatty acids → milk fat.
Minerals (Ca²⁺, Cl⁻, etc.) → micelle structure.

Transport flows:

Nutrients enter from blood → interstitial fluid → epithelial cells.
Cells assemble:
- Lactose in Golgi
- Caseins in ER/Golgi
- Milk fat globules in cytoplasm
- Then secrete into the alveolar lumen.

📌 For culture: media must supply correct substrates + hormonal triggers.

🧫 6. Cell Extraction for Cellular Agriculture

Process:

Choose cow with optimal physiological stage.
Transport mammary gland (~25 kg) from slaughterhouse aseptically.
Remove fat and connective tissue.
Excise parenchymal tissue containing milk-producing epithelial cells.
Bring to lab for digestion, isolation, and culture.

Key challenges:

Location inside gland varies; difficult to standardize sampling.
Need to preserve viability and sterility.

🎯 Final Takeaways

Plant-based drinks

More climate-friendly.
Not nutritionally equivalent to milk.
Limited bioactivity → no substitute for milk’s physiological effects.

Precision fermentation

Can make specific milk proteins.
But cannot replicate the complexity of real milk.

Cell-based milk

Most biologically faithful approach.
Requires deep understanding of mammary physiology, hormones & milk synthesis.
Still in early stages, but promising for 2030+.

Quiz

Score: 0/30 (0%)

Q0. Why does methane reduction play a central role in Denmark’s climate goals related to dairy production?

Methane has a shorter atmospheric lifespan than CO₂ and is therefore less important

Methane is primarily emitted by plant-based agriculture

Methane is far more potent than CO₂ and cows produce most of it in Denmark

Methane emissions come mainly from industrial processes rather than agriculture

Q1. What is a major limitation of plant-based drinks compared to cow’s milk?

They contain too much lactose

They lack equivalent protein levels, amino acid quality, and bioactive components

They require refrigeration while milk does not

They cannot be digested by humans

Q2. In the cell viability experiment, why was a whey-like phase prepared from each drink sample?

To remove nutrients so that cells are not overstimulated

To equalize fat and protein interference and isolate bioactive soluble components

To sterilize the samples chemically

To prevent sugars from affecting osmotic pressure

Q3. Which plant-based drink showed the most similar effect to cow’s milk on intestinal cell viability?

Soy

Oat

Spelt

Rice

Q4. What cellular assay was used to measure growth/viability in the lecture's experiment?

MTT assay

Trypan blue exclusion

Resazurin/Alamar Blue assay

LDH release assay

Q5. Why can’t precision fermentation products currently replicate real milk?

Because microbes cannot produce any milk proteins

Because milk contains over 2,000 components, far beyond the limited protein sets produced

Because fermentation technology is illegal

Because fermented proteins degrade instantly in solution

Q6. Which element of milk synthesis primarily depends on glucose availability?

Milk fat globules

Casein micelles

Lactose production

Mineral concentration

Q7. What happens to the mammary gland of a heifer when the ovaries are removed?

It becomes larger and more glandular

It becomes dominated by fat tissue with little parenchyma

It produces milk spontaneously

It increases its duct size but not alveoli

Q8. Why is physiological stage of a cow important when harvesting mammary tissue for cell culture?

Mammary cells change color depending on stage

Only lactating animals have any mammary epithelium

Hormonal and structural states strongly influence the type and readiness of milk-producing cells

Non-lactating cows have toxic metabolites in mammary tissue

Q9. Why must casein be removed before adding milk samples to cell cultures?

Casein prevents medium buffering

Casein causes protein-shock toxicity at high concentration

Casein binds to plastic surfaces and eliminates other nutrients

Casein inhibits mitochondrial respiration

Q10. Which hormone is most important for ductal and alveolar mammary gland development during puberty?

Prolactin

Estrogen

Progesterone

Insulin

Q11. What is the main function of mammary alveoli in milk production?

To transport milk to the cistern

To synthesize and secrete milk components into the lumen

To filter pathogens from blood

To store fat globules for later release

Q12. What is the significance of the 305-day lactation standard in dairy production?

It corresponds to the natural maximum length of lactation

It is an industry-standardized period used for comparing milk yields

It is the time needed for milk to reach nutritional maturity

It equals the cow’s gestation period

Q13. Why is cell-based milk conceptually more complete than precision fermentation?

Cells are easier to grow than microbes

Mammary cells inherently contain the full genetic machinery to produce nearly all natural milk components

Cells do not require any nutrients

Cells cannot produce hormones or bioactives

Q14. Which step is critical for ensuring sterility when collecting mammary tissue at slaughter?

Flushing the udder with water

Keeping the dissection aseptic after cutting through the skin

Heating the tissue to denature microbes

Rinsing tissue in ethanol

Q15. True or False: Plant-based drinks tested in the experiment generally increased intestinal cell growth similarly to cow’s milk.

True

False

Q16. True or False: Perfect Day’s recombinant beverages contain thousands of milk components similar to natural milk.

True

False

Q17. True or False: Casein removal is necessary because intact milk proteins cause osmotic swelling of cultured mammalian cells.

True

False

Q18. True or False: Mammary tissue from anywhere in the udder is equivalent for cell isolation purposes.

True

False

Q19. True or False: The lecture emphasizes that plant-based drinks are nutritionally identical substitutes for cow’s milk.

True

False

Q20. True or False: Milk contains growth factors that stimulate intestinal cell proliferation.

True

False

Q21. True or False: Mammary epithelial cells secrete milk into the duct system, which transports it to the cisternal region.

True

False

Q22. True or False: Removing ovaries increases mammary parenchymal development because estrogen inhibition is relieved.

True

False

Q23. True or False: Glucose is the primary precursor for lactose synthesis in milk production.

True

False

Q24. True or False: Growth hormone contributes to the expansion of mammary parenchymal tissue.

True

False

Q25. True or False: The lecture states that cow’s milk contains fewer components than plant-based drinks.

True

False

Q26. True or False: Alveoli regress to a near-prepubertal state during involution.

True

False

Q27. True or False: Precision-fermented milk already includes hormones and bioactive molecules identical to cow's milk.

True

False

Q28. True or False: In the experiment, soy-based drinks consistently increased cell viability across all tested concentrations.

True

False

Q29. True or False: Mammary cells require specific hormonal cues in vitro to initiate milk secretion.

True

False